Factor Xa inhibitors

ABSTRACT

The invention provides compounds which specifically inhibit factor Xa activity having the structure A 1 —A 2 —(A 3 ) m —B, where m is 0 or 1. A compound of the invention is characterized, in part, in that it exhibits a specific inhibition of factor Xa activity with a K i ≦100 μM, preferably ≦2 nM, and does not substantially inhibit the activity of other proteases involved in the coagulation cascade. The invention further provides methods of specifically inhibiting the activity of factor Xa and of inhibiting blood clotting in vitro and in an individual and methods of detecting factor Xa levels or activity.

The present application is a continuation-in-part of U.S. Ser. No.08/947,794 filed Oct. 8, 1997 and issued as U.S. Pat. No. 5,849,510,which is a continuation of prior application Ser. No. 08/428,404, filedApr. 25, 1995 now abandoned, which is a continuation-in-part of priorapplication Ser. No. 08/233,054, filed Apr. 26, 1994 now abandoned, allof which are incorporated herein by reference.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The present invention relates generally to the inhibition of bloodclotting proteins and more particularly to specific inhibitors of theblood clotting enzyme factor Xa.

BACKGROUND INFORMATION

The ability to form blood clots is vital to survival. In certain diseasestates, however, the formation of blood clots within the circulatorysystem is itself a source of morbidity. Thus, it sometimes can bedesirable to prevent blood clot formation. However, it is not desirableto completely inhibit the clotting system because life threateninghemorrhage would ensue.

In order to reduce the intravascular formation of blood clots, thoseskilled in the art have endeavored to develop an effective inhibitor ofprothrombinase or of factor Xa, which is incorporated into theprothrombinase complex where it activates thrombin during clotformation.

Appropriate concentrations of a factor Xa inhibitor would increase thelevel of prothrombinase forming agents required to initiate clotting butwould not unduly prolong the clotting process once a thresholdconcentration of thrombin had been obtained. However, despite the longstanding recognition of the desirability of such an inhibitor, there isat present no effective, specific factor Xa inhibitor in clinical use.

In many clinical applications there is a great need for anti-coagulanttreatment. The currently available drugs are not satisfactory in manyspecific clinical applications. For example, nearly 50% of patients whoundergo a total hip replacement develop deep vein thrombosis (DVT). Thecurrently approved therapies include fixed dose low molecular weightheparin (LMWH) and variable dose heparin. Even with these drug regimes,10% to 20% of patients develop DVT and 5% to 10% develop bleedingcomplications.

Another clinical situation for which better anti-coagulants are neededconcerns subjects undergoing transluminal coronary angioplasty and atrisk for myocardial infarction or suffering from crescendo angina. Thepresent, conventionally accepted therapy, which consists ofadministering heparin and aspirin, is associated with a 6% to 8% abruptvessel closure rate within 24 hours of the procedure. The rate ofbleeding complications requiring transfusion therapy due to the use ofheparin also is approximately 7%. Moreover, even though delayed closuresare significant, administration of heparin after the termination of theprocedures is of little value and can be detrimental.

The most widely used blood-clotting inhibitors are heparin and therelated sulfated polysaccharides, LMWH and heparin sulfate. Thesemolecules exert their anti-clotting effects by promoting the binding ofa natural regulator of the clotting process, anti-thrombin III, tothrombin and to factor Xa. The inhibitory activity of heparin primarilyis directed toward thrombin, which is inactivated approximately 100times faster than factor Xa. Although relative to heparin, heparinsulfate and LMWH are somewhat more potent inhibitors of Xa than ofthrombin, the differences in vitro are modest (3-30 fold) and effects invivo can be inconsequential. Hirudin and hirulog are two additionalthrombin-specific anticoagulants presently in clinical trials. However,these anticoagulants, which inhibit thrombin, also are associated withbleeding complications.

Preclinical studies in baboons and dogs have shown that specificinhibitors of factor Xa prevent clot formation without producing thebleeding side effects observed with direct thrombin inhibitors. Suchfactor Xa inhibitors include, for example, 2,7-bis-(4-amidinobenzylidene)-cycloheptanone and Nα-tosylglycyl-3-amidinophenylalaninemethyl ester (“TENSTOP”), which have effective inhibitory concentrations(K_(i)'s) of about 20 nM and 800 nM, respectively.(+)-(2S)-2-(4({(3S)-1-acetimidoyl-3-pyrrolidinyl}oxy)phenyl)-3-(7-amidino-2-naphthyl)propanoicacid also is representative of a class of factor Xa inhibitors (Katakuraet al., Biochem. Biophys. Res. Comm. 197:965-972 (1993)). Thus far,however, these compounds have not been developed clinically.

Specific protein inhibitors of factor Xa also have been identified andinclude, for example, antistasin (“ATS”) and tick anticoagulant peptide(“TAP”). ATS, which isolated from the leech, Haementerin officinalis,contains 119 amino acids and has a K_(i) for factor Xa of 0.05 nM. TAP,which is isolated from the tick, Ornithodoros moubata, contains 60 aminoacids and has a K_(i) for factor Xa of about 0.5 nM.

The effectiveness of recombinantly-produced ATS and TAP have beeninvestigated in a number of animal model systems. Both inhibitorsdecrease bleeding time compared to other anticoagulants and preventclotting in a thromboplastin-induced, ligated jugular vein model of deepvein thrombosis. The results achieved in this model correlate withresults obtained using the current drug of choice, heparin.

Subcutaneous ATS also was found to be an effective treatment in athromboplastin-induced model of disseminated intravascular coagulation(DIC). TAP effectively prevents “high-shear” arterial thrombosis and“reduced flow” caused by the surgical placement of a polyester(“DACRON”) graft at levels that produced a clinically acceptableprolongation of the activated partial thromboplastin time (aPTT), i.e.,less than about two fold prolongation. By comparison, standard heparin,even at doses causing a five fold increase in the aPTT, did not preventthrombosis and reduced flow within the graft. The aPTT is a clinicalassay of coagulation which is particularly sensitive to thrombininhibitors.

ATS and TAP have not been developed clinically. One major disadvantageof these two inhibitors is that administration of the required repeateddoses causes the generation of neutralizing antibodies, thus limitingtheir potential clinical use. Moreover, the sizes of TAP and ATS renderoral administration impossible, further restricting the number ofpatients able to benefit from these agents.

A specific inhibitor of factor Xa would have substantial practical valuein the practice of medicine. In particular, a factor Xa inhibitor wouldbe effective under circumstances where the present drugs of choice,heparin and related sulfated polysaccharides, are ineffective or onlymarginally effective. Thus, there exists a need for a low molecularweight, factor Xa-specific blood clotting inhibitor that is effective,but does not cause unwanted side effects. The present inventionsatisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides compounds that specifically inhibitfactor Xa activity. A compound of the invention has the structureX₁-Y-I-R-X₂, wherein X₁ is a hydrogen (H), acyl, alkyl or arylalkylgroup, or one or more amino acids, and X₂ is a modified C-terminalgroup, one or more carboxy-protecting groups (see below), one or moreamino acids, or other substituents, and Y, I and R refer to the aminoacids tyrosine, isoleucine and arginine, respectively, and topeptidomimetic or organic structures that have the same functionalactivities as tyrosine, isoleucine and arginine, respectively. Inaddition, a compound of the invention has the structure,A1—A2—(A3)_(m)—B, as defined herein.

A compound of the invention can be linear or cyclic, between about 2 and43 residues in length and modified at the N-terminus or C-terminus orboth. Such compounds exhibit a specific inhibition of factor Xa activitywith a K_(i)≦100 μM, preferably a K_(i)≦2 nM, and do not substantiallyinhibit the activity of other proteases involved in the coagulationcascade. Specific examples of such compounds includeAc-Tyr-Ile-Arg-Leu-Ala-NH₂(SEQ ID NO: 1); Ac-Tyr-Ile-Arg-Leu-Pro-NH₂(SEQID NO: 2); Ac-(iBu)Tyr-Ile-Arg-Leu-Pro-NH₂(SEQ ID NO: 3);Ac-Tyr-Ile-Arg-N(CH₃)O(CH₃); Ac-Tyr-{Ψ(CH₂NH)}-Ile-Arg-Leu-Pro-NH₂(SEQID NO: 4) (where “Ψ” indicates a pseudo peptide bond, which, forexample, can be a reduced bond as indicated by “(CH₂NH)”; pseudo peptidebonds are indicated by “Ψ” enclosed in brackets, “{Ψ}”);Ac-Tyr-Ile-Arg-NH-CH₂(4-Pyridyl);Ac-Tyr-Ile-{Ψ(CH₂NH)}-Arg-Leu-Pro-NH₂(SEQ ID NO: 5);Ac-Tyr-Chg-Arg(NO₂)-{Ψ(CH₂NH)}-Leu-NH₂;Ac-Tyr-Ile-Arg-{Ψ(COCH₂)}-Gly-Pro-NH₂(SEQ ID NO: 6);Ac-Tyr-Ile-Dab(N^(γ)—C₃H₇N)-Leu-Ala-NH₂(SEQ ID NO: 7);Ac-Tyr-Ile-PalMe(3)-NH₂; Tyr-Ile-Arg-NH₂; (D)-Tyr-Ile-Arg-Leu-Pro-NH₂;Ac-(Bzl)Gly-(Chx)Gly-(3-guanidopropyl)Gly-NH₂;Cyclo(Gly-Tyr-Ile-Arg-Gly) (SEQ ID NO: 8);Tfa-(iBu)Tyr-Chg-Arg-Leu-Pro-NH₂; Ac-pAph-Chg-Arg-Leu-Pro-NH₂;Ac-Nal(2)-Chg-Arg-Leu-Pro-NH₂; Ac-pAph-Chg-PalMe(3)-NH₂; andpharmaceutically acceptable salts and C-terminal derivatives such asamides, esters, alcohols and aldehydes thereof (see, also, Table 5).Methods of specifically inhibiting the activity of factor Xa and ofinhibiting blood-clotting in an individual also are provided. Methods ofdetecting factor Xa levels or activity are provided as well.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the blood coagulation cascade.

FIG. 2 exemplifies a structure for a compound of the invention.

FIG. 3 shown a synthesis scheme for preparing some compounds of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

Blood coagulation is a complex process involving a progressivelyamplified series of enzyme activation reactions in which plasma zymogensare sequentially activated by limited proteolysis. Mechanistically theblood coagulation cascade has been divided into intrinsic and extrinsicpathways, which converge at the activation of factor X; subsequentgeneration of thrombin proceeds through a single common pathway (seeFIG. 1).

Present evidence suggests that the intrinsic pathway plays an importantrole in the maintenance and growth of fibrin formation, while theextrinsic pathway is critical in the initiation phase of bloodcoagulation. It is generally accepted that blood coagulation isphysically initiated upon formation of a tissue factor/factor VIIacomplex. Once formed, this complex rapidly initiates coagulation byactivating factors IX and X. The newly generated factor Xa then forms aone-to-one complex with factor Va and phospholipids to form aprothrombinase complex, which is responsible for converting solublefibrinogen to insoluble fibrin. As time progresses, the activity of thefactor VIIa/tissue factor complex (extrinsic pathway) is suppressed by aKunitz-type protease inhibitor protein, tissue factor pathway inhibitor(TFPI), which, when complexed to factor Xa, can directly inhibit theproteolytic activity of factor VIIa/tissue factor. In order to maintainthe coagulation process in the presence of an inhibited extrinsicsystem, additional factor Xa is produced via the thrombin-mediatedactivity of the intrinsic pathway. Thus, thrombin plays a dualautocatalytic role, mediating its own production and the conversion offibrinogen to fibrin.

The autocatalytic nature of thrombin generation is an importantsafeguard against uncontrolled bleeding and it ensures that, once agiven threshold level of prothrombinase is present, blood coagulationwill proceed to completion, effecting, for example, an end of thehemorrhage. Thus, it is most desirable to develop agents that inhibitcoagulation without directly inhibiting thrombin.

The present invention provides YIR peptides, which are compounds thatinhibit factor Xa activity but do not substantially inhibit the activityof other proteases involved in the blood coagulation pathway. As usedherein, the term “compound” or “YIR peptide” refers to a non-naturallyoccurring Tyr-Ile-Arg (YIR) peptide and analogues and mimetics thereof,which can inhibit factor Xa activity. The YIR sequence, itself, isreferred to herein as the “YIR motif” and consists of the tripeptidetyrosine-isoleucine-arginine or a functional equivalent thereof such aspAph-Chg-PalMe(3), pAph-Chg-PalMe(3)-NH₂ and pAph-Chg-AMP(4) (see Table1 for abbreviations). Such compounds of the invention contain at leastone YIR motif or a functional equivalent thereof and are capable ofspecifically inhibiting the activity of factor Xa. For convenience, theterms “compound” and “YIR peptide” are used broadly herein to refer tothe peptides of the invention, including functional equivalents such aspeptide analogs, peptide mimetics and synthetic organic compounds. Afunction equivalent of a YIR peptide of the invention can becharacterized, in part, by having a structure as disclosed herein and byhaving a K_(i)≦100 μM for inhibiting factor Xa activity (see ExampleXXXVII).

Peptide analogs of a YIR peptide of the invention include, for example,peptides containing non-naturally occurring amino acids or chemicallymodified amino acids, provided the compound retains factor Xa inhibitoryactivity (see, for example, Table 2). Similarly, peptide mimetics arenon-amino acid chemical structures that mimic the structure of a YIRpeptide of the invention and retain factor Xa inhibitor activity. Suchmimetics are characterized generally as exhibiting similar physicalcharacteristics such as size, charge or hydrophobicity that is presentedin the appropriate spatial orientation as found in the normal YIRpeptide counterpart. A specific example of a peptide mimetic is acompound in which the amide bond between one or more of the amino acidsis replaced, for example, by a carbon-carbon bond or other bond as iswell known in the art (see, for example, Sawyer, in Peptide Based DrugDesign pp. 387-422 (ACS, Washington D.C. 1995), which is incorporatedherein by reference). Thus, the invention further provides factor Xainhibitory compounds having the structure A1—A2—(A3)_(m)—B, where m is 0or 1, as disclosed herein (see below). Examples of such peptides, whichcan be mimetic compounds, are provided herein.

As used herein, the term “amino acid” is used in its broadest sense tomean the twenty naturally occurring amino acids, which are translatedfrom the genetic code and comprise the building blocks of proteins,including, unless specifically stated otherwise, L-amino acids andD-amino acids, as well as chemically modified amino acids such as aminoacid analogs, naturally-occurring amino acids that are not usuallyincorporated into proteins such as norleucine, and chemicallysynthesized compounds having properties known in the art to becharacteristic of an amino acid. For example, analogs or mimetics ofphenylalanine or proline, which allow the same conformationalrestriction of the peptide compounds as natural Phe or Pro, are includedwithin the definition of “amino acids” and are known to those skilled inthe art. Such analogs and mimetics are referred to herein as “functionalequivalents” of an amino acid. Other examples of amino acids and aminoacids analogs are listed by Roberts and Vellaccio (The Peptides:Analysis, Synthesis, Biology, Eds. Gross and Meienhofer, Vol. 5, p. 341,Academic Press, Inc., N.Y. 1983, which is incorporated herein byreference). Abbreviations of amino acids, amino acid analogs and mimeticstructures are listed in Table 1.

TABLE 1 Abbreviations used in the specification Compound Abbreviation #1Abbreviation #2* Acetyl Ac Alanine Ala A 3-(2-Thiazolyl)-L-alanine TzaAmidine AMDN Amidoxime (C(NH₂)═N—OH) (CNOH.NH₂) (N-methylpyridinium) AMPmethyl (4-(N-methylpyridinium)) AMP(4) methyl 1-(N-Methylpyridinium)AEMP ethy-1-yl 1-(4-(N-methylpyridinium)) AEMP(4) eth-1-yl Arginine ArgR Asparagine Asn N Aspartic acid Asp D Benzoyl Bz 2-BenzofuranecarboxyBzf Benzyl Bzl Benzyloxycarbonyl Cbz 5-Benzimidazolecarboxy 5-Bzimt-Butyloxycarbonyl Boc Benzotriazol-1-yloxy Bop tris-(dimethylamino)-phosphonium- hexafluorophosphate β-Alanine βAla β-Valine βValβ-(2-Pyridyl)-alanine Pal (2) β-(3-Pyridyl)-alanine Pal (3)β-(4-Pyridyl)-alanine Pal (4) β-(3-N-Methylpyridinium)- PalMe (3)alanine Bromo-tris-pyrrolidino- PyBroP phosphonium- hexafluorophosphatet-Butyl tBu, But t-Butyloxycarbonyl Boc Caffeic acid CaffCarbonyldiimidazole CDI Cysteine Cys C 5-chloroindole-2-carboxy CICACyclohexyl Chx Cyclohexylalanine Cha Cyclohexylglycine Chg2,4-Diaminobutyric acid Dab Dab-derived Dab(N^(γ)—C₃H₇N)dimethylamidinium 2,3-Diaminopropionic acid Dap Dap-derivedDap(N^(β)—C₃H₇N) dimethylamidinium 3,5-Dinitrotyrosine Tyr(3,5-NO₂)Y(3,5-NO₂) 3,5-Diiodotyrosine Tyr(3,5-I) Y(3,5-I) 3,5-DibromotyrosineTyr(3,5-Br) Y(3,5-Br) N,N-diisobutylcarboxamide DIBAN,N-diisopropylcarboxamide DIPA 4-N,N-Dimethylamino DMAP pyridine9-Fluorenylmethyloxy- Fmoc carbonyl 5-Fluoroindole-2-carboxy FICA FormylFor Glutamine Gln Q Glutamic acid Glu E y-Carboxyglutamic acid GlaGlycine Gly G Histidine His H Homoarginine hArg hR 5-Hydroxyindole-5-Hic 2-carboxy N-Hydroxybenzotriazole HOBt 3-Hydroxyproline HypIminodiacetic acid Ida 5-aminoindole-2-carboxy 5AM2IN5-nitroindole-2-carboxy 5NOINDC DL-Indoline-2-carboxy 2INCA Isobutyl iBuIsoleucine Ile I Isonicotinic acid Isn N-Methyl-isnicotinic acid IsnMeIsonipecotic acid Ina Isopropanol iPrOH 1-Isoquinolinecarboxy 1-Iqc3-Isoquinolinecarboxy 3-Iqc Leucine Leu L tert-Leucine Tle Lysine Lys KMercapto-β,β- Mpp cyclopentamethylene- propionic acid Mercaptoaceticacid Mpa Mercaptopropionic acid Mpr Methanol MeOH Methionine Met M4-Morpholinocarbonylamide MORA N-methylmorpholine NMM 1-NaphthylalanineNal(1) 2-Naphthylalanine Nal(2) Nicotinic acid Nic Nipecotic acid NpaN-methyl nicotinic acid NicMe Norarginine nArg nR Norleucine Nle nLNorvaline Nva nV Ornithine Orn Ornithine-derived Orn(N^(δ)—C₃H₇N)dimethylamidinium Phenyl Ph Phenylalanine Phe F p-GuanidinophenylalaninePhe(Gua) F(pGua) p-Aminophenylalanine Phe(NH₂) F(pNH₂)p-Chlorophenylalanine Phe(Cl) F(pCl) p-Flurophenylalanine Phe(F) F(pF)p-Nitrophenylalanine Phe(NO₂) F(pNO₂) p-Hydroxyphenylglycine Pgl(OH)p-Toluenesulfonyl Tos 2,2,5,7,8-Pentamethyl- Pmc chroman-6-sulfonylm-Amidinophenylalanine mAph p-Amidinophenylalanine pAph PhenylglycinePgl Phenylmalonic acid Pma Piperidinyl PIP 1-Piperidinocarbonyl PIPAamide L-Pipecolonic acid Pip Proline Pro P 2-Pyrazinecarboxy Pza2-Quinolinecarboxy 2-Qca 4-Quinolinecarboxy 4-Qca Sarcosine SarS-tert-butyl SBut SCAL linker attached to SCAL-TG “TENTAGEL” Serine SerS Tetrahydroisoquinoline- Tic 3-carboxyl Threonine Thr T TrifluoroacetylTfa Tryptophan Trp W Tyrosine Tyr Y 3-iodotyrosine Tyr(3-I) Y(3-I)O-Methyl tyrosine Tyr(Me) Y(Me) Valine Val V *Amino acids of Dconfiguration are denoted either by D-prefix using three-letter code(eg., D-Ala, D-Cys, D-Asp, D-Trp) or with lower case letters using theone-letter code (a, c, d, w, respectively).

As used herein, the term “factor Xa activity” refers to the ability offactor Xa, by itself or in the assembly of subunits known as theprothrombinase complex, to catalyze the conversion of prothrombin tothrombin. When used in reference to factor Xa activity, the term“inhibition” includes both the direct and the indirect inhibition offactor Xa activity. Direct inhibition of factor Xa activity can beaccomplished, for example, by the binding of a YIR peptide of theinvention to factor Xa or to prothrombinase so as to prevent the bindingof prothrombin to the prothrombinase complex active site. Indirectinhibition of factor Xa activity can be accomplished, for example, bythe binding of a compound of the invention to soluble factor Xa so as toprevent its assembly into the prothrombinase complex.

As used herein, the term “specific” when used in reference to theinhibition of factor Xa activity means that a YIR peptide can inhibitfactor Xa activity without substantially inhibiting the activity ofother specified proteases, including plasmin and thrombin (using thesame concentration of the inhibitor). Such proteases are involved in theblood coagulation and fibrinolysis cascade (see Table 2; see, also,Example XXVII).

TABLE 2 Inhibitory activities of selected compounds against five enzymesKi (μM) Compound Xa Thrombin Plasmin Trypsin Elastase Ac-Y-I-R-L-A- 1.5100 NT >200* >100 A-E-T (SEQ ID NO:9) Ac-Y-I-R-L-P0.5 >200 >200 >200 >100 (SEQ ID NO:10) y-I-R-L-P 0.2 >200 >200 >200 >100Ac-(iBu)Y-I- 0.04 25 >200 NT >100 R-L-P (SEQ ID NO:11) “TENSTOP” 22 >200 NT >200 *Indicates that there was no significant inhibition ofenzyme activity at the highest concentration of compound (indicated)tested.

The results in Table 2 demonstrate that the YIR peptides of theinvention are useful as inhibitors of factor Xa but do not substantiallyinhibit the activity of other serine proteases such as thrombin orplasmin, which are involved in the process of blood coagulation andfibrinolysis.

As used herein, the term “substituent” refers to any of various chemicalgroups that is substituted onto the peptide backbone or side chain of apeptide, peptide analogue, mimetic or organic compound disclosed herein.A substituent can include any of a variety of different moieties knownto those skilled in the art (see, for example, Giannis and Kolter,Angew. Chem. Int. Ed. Engl. 32:1244-1267 (1993), which is incorporatedherein by reference). Numerous examples demonstrating the substitutionof a substituent are disclosed herein including, for example,substitution of a pNH₂ substituent onto phenylalanine to obtain F(pNH₂)and the substitution of a halogen onto a tyrosine to obtain, for exampleY(3-I) or Y(3,5-I). In addition, a substituent can be, for example, aheteroatom such as nitrogen (N; see, for example, Pal), oxygen (O; see,for example, O-methyltyrosine) or sulfur (S; see, for example,Tyr(SO₃H)), which can be substituted with a substituent. Thus, an N-, S-or O-containing moiety such as —SO₃H is considered a “substituent” asdefined herein. Furthermore, a substituent can be an amino-protectinggroup or carboxy-protecting group.

As used herein, the term “alkyl” is used in the broadest sense to meansaturated or unsaturated, linear, branched or cyclic chains of about 1to 13 carbon atoms. Thus, the term “alkyl” includes, for example,methyl, ethyl, n-propyl, isopropyl, sec-butyl, 1-methylbutyl,2,2-dimethylbutyl, 2-methylpentyl, 2,2-dimethylpropyl, n-pentyl andn-hexyl groups, alkylene groups, cyclic chains of carbon atoms suchcyclohexyl and cyclopentyl groups, as well as combinations of linear orbranched chains and cyclic chains of carbon atoms such as amethyl-cyclohexyl or cyclopropyl-methylene group. In addition, it shouldbe recognized that an alkyl as defined herein can be substituted with asubstituent. Similarly, the term “acyl” is used in its broadest sense tomean saturated or unsaturated, linear, branched or cyclic chains ofabout 1 to 13 carbon atoms, which contain a carboxyl group. Thus, theterm “acyl” encompasses, for example, groups such as formyl, acetyl,benzoyl and the like.

The term “aryl” refers to aromatic groups containing about 5 to 13carbon atoms and at least one “ring” group having a conjugated pielectron system. Examples of aryls include, for example, heterocyclicaryl groups, biaryl groups, and analogues and derivatives thereof, allof which optionally can be substituted with one or more substituents.The term “arylalkyl” refers to an alkyl as defined above substitutedwith an aryl group. Suitable arylalkyl groups include benzyl, picolyland the like, all of which optionally can be substituted.

The terms “heteroalkyl,” “heteroarylalkyl” and “heteroaryl” also areused herein and refer to an alkyl, an arylalkyl and an aryl,respectively, that is substituted with one or more heteroatoms such as aN, O or S atom. In addition, the term “heterocyclic” is used inreference to a cyclic alkyl or an aryl group that is substituted withone or more heteroatoms. Numerous examples of heteroalkyls,heteroarylalkyls, heteroaryls and heterocyclics are disclosed, forexample, in Tables 1 and 3, or are otherwise known in the art.

The peptides of the invention can be modified at the N-terminus or theC-terminus using an amino-protecting group or carboxy-protecting group,respectively. Numerous such modifications are disclosed herein (see, forexample, Table 3). The N-terminus of a peptide or peptide analog can bechemically modified such that the N-terminus amino group is substituted,for example, by an acetyl, cyclopentylcarboxy, isoquinolylcarboxy,furoyl, tosyl, pyrazinecarboxy or other such group, which can besubstituted by a substituent as described above. The N-terminal aminogroup also can be substituted, for example, with a reverse amide bond.It should be recognized that the term “amino group” is used broadlyherein to refer to any free amino group, including a primary, secondaryor tertiary amino group, present in a peptide. In comparison, the term“N-terminus” refers to the α-amino group of the first amino acid presentin a peptide written in the conventional manner.

The N-terminus of a peptide of the invention can be protected by linkingthereto an amino-protecting group. The term “amino-protecting group” isused broadly herein to refer to a chemical group that can react with afree amino group, including, for example, the α-amino group present atthe N-terminus of a peptide of the invention. By virtue of reactingtherewith, an amino-protecting group protects the otherwise reactiveamino group against undesirable reactions as can occur, for example,during a synthetic procedure or due to exopeptidase activity on a finalcompound. Modification of an amino group also can provide additionaladvantages, including, for example, increasing the solubility or theactivity of the compound. Various amino-protecting groups are disclosedherein (see Table 3) or otherwise known in the art and include, forexample, acyl groups such as an acetyl, picoloyl, tert-butylacetyl,tert-butyloxycarbonyl, benzyloxycarbonyl, benzoyl groups, including, forexample, a benzyloxime such as a 2-aryl-2-O-benzyloxime (see ExampleXVI), as well as an aminoacyl residue, which itself can be modified byan amino-protecting group. Other amino-protecting groups are described,for example, in The Peptides, eds. Gross and Meienhofer, Vol. 3(Academic Press, Inc., N.Y. 1981); and by Greene and Wuts, in ProtectiveGroups in Organic Synthesis 2d ed., pages 309-405 (John Wiley & Sons,New York (1991), each of which is incorporated herein by reference. Theproduct of any such modification of the N-terminus amino group of apeptide or peptide analog of the invention is referred to herein as an“N-terminal derivative.”

Similarly, a carboxy group such as the carboxy group present at theC-terminus of a peptide can be chemically modified using acarboxy-protecting group. The terms “carboxy group” and “C-terminus” areused in a manner consistent with the terms “amino group” and“N-terminus” as defined above. A carboxy group such as that present atthe C-terminus of a peptide can be modified by reduction of theC-terminus carboxy group to an alcohol or aldehyde or by formation of anoral ester or by substitution of the carboxy group with a substituentsuch as a thiazolyl, cyclohexyl or other group. Oral esters are wellknown in the art and include, for example, alkoxymethyl groups such asmethoxymethyl, ethoxymethyl, iso-propoxymethyl and the like; the α-(C₁to C₄)alkoxyethyl groups such as methoxyethyl, ethoxyethyl,propoxyethyl, isopropoxyethyl and the like; the2-oxo-1,3-dioxolen-4-ylmethyl groups such as5-methyl-2-oxo-1,3-dioxolen-4-ylmethyl,5-phenyl-2-oxo-1,3-dioxolen-4-ylmethyl and the like; the C₁ to C₃alkylthiomethyl groups such as methylthiomethyl, ethylthiomethyl,isopropylthiomethyl and the like; the acyloxymethyl groups such aspivaloyloxymethyl, α-acetoxymethyl and the like; theethoxycarbonyl-1-methyl group; the α-acyloxy-α-substituted methyl groupssuch as α-acetoxyethyl, the 3-phthalidyl or 5,6-dimethylphthalidylgroups, the 1-(C₁ to C₄ alkyloxycarbonyloxy)eth-1-yl groups such as the1-(ethoxycarbonyloxy)eth-1-yl group; and the 1-(C₁ to C₄alkylaminocarbonyloxy)eth-1-yl group such as the1-(methylaminocarbonyloxy)eth-1-yl group.

A peptide of the invention can be modified by linking thereto acarboxy-protecting group. Carboxy-protecting groups are well known inthe art and, by virtue of being bound to a peptide, protect a carboxygroup against undesirable reactions (see, for example, Greene and Wuts,supra, pages 224-276 (1991), which is incorporated herein by reference).The skilled artisan would recognize that such modifications as describedabove, which can be effected upon the N-terminus amino group orC-terminus carboxy group of a peptide, similarly can be effected uponany reactive amino group or carboxy group present, for example, on aside chain of an amino acid or amino acid analog in a peptide of theinvention. Methods for performing such modifications are disclosedherein or otherwise known in the art.

The present invention provides compounds that specifically inhibitfactor Xa activity. A compound of the invention has the generalstructure X₁-YIR-X₂ or a functional equivalent thereof, wherein X₁ is anH, acyl, alkyl, arylalkyl, or one or more amino acids, and X₂ is amodified C-terminal group, one or more carboxy-protecting groups, or oneor more amino acids or other substituent such as an amino-protectinggroup. A compound of the invention is useful as an anticoagulant fortherapeutic treatment of a variety of clinical conditions. A compound ofthe invention also is useful in a variety of laboratory procedures toprevent the clotting of blood samples.

The invention also provides a compound that specifically inhibits theactivity of factor Xa, having the general formula A1—A2—(A3)_(m)—B,wherein m is 0 or 1 and A1 is R₁—R₂—R₃, A2 is R₄—R₅—R₆ and A3 isR₇—R₈—R₉; wherein R₁ is selected from the group consisting of 1 to 20amino acids;

X is selected from the group consisting of N, CH and NC(O), and R′₁ andR″₁ independently are selected from the group consisting of H, alkyl,acyl, aryl, arylalkyl and an amino-protecting group and wherein R₁ canbe substituted by a substituent; R₂ is —CR₉₉R₁₀₀—, wherein R₉₉ and R₁₀₀independently are selected from the group consisting of an H; alkyl,arylalkyl, heteroarylalkyl and heteroaryl, and wherein R₉₉ and R₁₀₀independently can be substituted with a substituent; R₃ is selected fromthe group consisting of —C(O)—, —CH₂—, —CHR₉₉—C(O)— and—C(O)—NR₃₅—CH₂—C(O)—, wherein R₃₅ is the CHR₅₅ group of the bridginggroup —C(O)—CR₅₅—; R₄ is selected from the group consisting of —CH₂— and—NR₅₀—, wherein R₅₀ is selected from the group consisting of H, alkyl,arylalkyl and heterocyclic; R₅ is >CR₂₀₁R₂₀₂, wherein R₂₀₁ and R₂₀₂independently are selected from the group consisting of H, alkyl, aryland arylalkyl, and wherein R₂₀₁ and R₂₀₂ independently can besubstituted with a substituent; R₆ is selected from the group consistingof —C(O)—, —CH₂— and —CHR₉₉—C(O)—; R₇ is selected from the groupconsisting of —CH₂— and —NR₅₁—, wherein R₅₁ is H, alkyl, arylalkyl,heteroalkyl and heteroarylalkyl, and any of these moieties substitutedby a substituent selected from the group consisting of Q and—(CH₂)_(n)-Q, wherein n is 1 to 5 and wherein Q is selected from thegroup consisting of an amino, amidino, imidazole and guanidino group,which can be substituted with a substituent, and a mono-, di-, tri- ortetra-alkylammonium of a pharmaceutically acceptable salt, isoureide orisothioureide thereof; R₈ is —CR₂₁₀R₂₁₁—, wherein R₂₁₀ and R₂₁₁independently are selected from the group consisting of H, alkyl,alkylaryl and heterocyclic, and any of these moieties substituted by asubstituent selected from the group consisting of Q and —(CH₂)_(n)-Q,wherein n is 1 to 5 and wherein Q is selected from the group consistingof an amino, amidino, imidazole and guanidino group, which can besubstituted with a substituent, and a mono-, di-, tri- ortetra-alkylammonium of a pharmaceutically acceptable salt, isoureide orisothioureide thereof; R₉ is selected from the group consisting of—C(O)—, —CH₂— and —CHR₉₉—C(O)—; and wherein, when m is 1, B is selectedfrom the group consisting of 1 to 20 amino acids, —NHR₅₂, —NR₆₀R₆₁,—OR₇₀ and —CHR₆₀R₆₁, wherein R₅₂ is selected from the group consistingof H, alkyl, arylalkyl, heteroarylalkyl and heteroaryl; wherein R₆₀ andR₆₁ independently are selected from the group consisting of H, alkyl,arylalkyl, aryl, heteroarylalkyl and heteroaryl, and wherein R₇₀ isselected from the group consisting of H, acyl, alkyl, arylalkyl andheteroarylalkyl, and wherein when m is 0, B is selected from the groupconsisting of 1 to 20 amino acids, —OR₇₀, —NHR₅₂ and —NR₆₀R₆₁, which isjoined to R₆ by an amide bond or an ester bond; wherein B can besubstituted with a substituent, provided that when R₃ is —CH₂— or—CHR₉₉—C(O)—, R₄ is NR₅₀; when R₄ is —CH₂—, R₃ is —C(O)— or—CHR₉₉—C(O)—; when R₄ is —CH₂—, R₃ is —C(O)— or —CHR₉₉—C(O)—; when R₆ is—CH₂—, R₇ is —NHR₅₁—; when R₇ is CH₂, R₆ is —C(O)— or —CHR₉₉—C(O)—; whenR₄ is —NR₅₀— and R₁ is

R₅₀ and R′₁ are taken together to form a bridging group having theformula: —C(O)—CHR₅₅—, wherein CHR₅₅ represents R₅₀ and the carbonylgroup represents R′₁, and R″₁, and R₅₅ independently are H, C₁ to C₆alkyl or arylalkyl; and when R₃ is —C(O)—NR₃₅—CH₂—C(O)—, then R₄ is—NR_(50—), R₁ is

R₃₅ and R′₁ are taken together to form a bridging group having theformula —C(O)CHR₅₅—, wherein C(O) represents R′₁ and CHR₅₅ representsR₃₅; R″₁ and R₅₅ independently are H or a C₁ to C₆ alkyl (see, forexample, FIG. 2).

A compound of the invention can contain a cyclic N-terminus formed byR₁, R₂, R₃ and, if desired, R₄. Such a compound is defined, for example,by the structure A1—A2—(A3)_(m)—B, as described above, wherein R₄ is—NR₅₀—, R₁ is

R₅₀ and R′₁ are taken together to form a bridging group of the formula—C(O)—CHR₅₅, wherein R₅₅ is H; R₁ is H or methyl; R₉₉ and R₁₀₀independently are selected from the group consisting of H, arylalkyl,alkyl and heteroalkyl or 1 to 3 carbon atoms, and wherein R₉₉ and R₁₀₀can be further linked to a moiety selected from the group consisting ofphenyl, thienyl, thiazolyl, pyridyl, naphthyl, thionaphthyl, indolyl orsaturated alkyl, alkoxy, monoalkylamino, dialkylamino,tetraalkylammonium, arylalkylamino, aminoalkylaryl, carboxy, halo,hydroxy, amino, amido, amidino, guanidino, triazolyl and sulfonyl, andR₃ is selected from the group consisting of —C(O)— and—C(O)—NR₃₅—CH₂—C(O)—.

Furthermore, in the compound A1—A2—(A3)_(m)—B, the R′1 and R″1 moietiescan be substituted with up to six substituents, including, for example,an alkyl, and optionally linked by a group such as —OCH₂—, —SCH₂—,>N—CH₂—, >NC(O)—, —CO— or —NY—CO—NZ, where Y and Z can be H, alkyl,arylalkyl or heteroarakyl. Moreover, R₉₉ and R₁₀₀ independently can besubstituted by a substituent such as a phenyl, thienyl, thiazolyl,pyridyl, naphthyl, thionaphthyl or indolyl group, or a saturated groupcorresponding thereto, optionally substituted by up to five groupsselected from alkyl, alkoxy, mono-, di- or tri-alkylamine,tetralkylammonium, arylalkylamino, aminoalkylaryl, carboxy, halogens,hydroxy, amino, amide, amidino, guanidino, triazolyl or sulfonyl. Apreferred compound with substitutions at the R₂ position is where R₁₀₀is an H and R₉₉ is either

where W in the substituted compound can be, for example, a halogen,hydroxyl, amino or amidino group, and J can be, for example, an O, S or—NR, where R is an H or an alkyl, aryl or arylalkyl.

A compound of the invention, which contains a substituent substituted onthe A2 moiety and exhibits factor Xa inhibitory activity, can have, forexample, the substitution of R₅₀, R₂₀₁ or R₂₀₂ with one or moreheteroatom substituents such as an N, O or S. R₂₀₂ also can besubstituted with a substituent selected from

Where X is a C, N or S; R is absent, H or alkyl, which can besubstituted with a heteroatom, and n is 1 to 5.

A compound of the invention, which contains a substituent substituted onthe A3 moiety and exhibits factor Xa inhibitory activity, can include,for example, the substitution of R₅₁ with one or more substituents suchas an H, alkyl, arylalkyl or heterocyclic, optionally substituted with aheteroatom such as an N, O or S. R₂₁₀ and R₂₁₁ can be, for example, thesubstituent —(CH₂)_(n)-Q, where n is about 1 to 5 and where Q is anamino, amidino, urea, imidazole, guanidino, mono-, di-, tri- ortetra-alkyl imminium of a pharmaceutically acceptable salt, isoureide orisothioureide. Alternatively, R₂₁₀ or R₂₁₁ can be, for example, analkyl, aryl or alkylaryl. These groups can be further substituted with asubstituent such as a hydroxy or C₁ to C₄ alkoxy group.

A compound of the invention can contain an alternative arrangement ofsubstituents comprising the B moiety. Such an alternative arrangement ofsubstituents can include, for example, the substitution of R₅₂ by an N,O or S or the substitution of R₆₀, R₆₁ or R₇₀ by one or more heteroatomsor alkyl groups.

The general structures disclosed herein represent the various compoundsof the invention, which retain factor Xa inhibitory activity such asimparted by the tripeptide, YIR. Also represented within the structuresdisclosed herein are compounds containing non-naturally occurring aminoacids, amino acid mimetics and other organic structures and substituentsexhibiting similar function. Such functional equivalents provide theappropriate spatial groupings of the desired charges and forces thatconfer effective factor Xa inhibitor function.

Specific examples of the compounds of the invention include, forexample, Ac-Tyr-Ile-Arg-Leu-Ala-NH₂; Ac-Tyr-Ile-Arg-Leu-Pro-NH₂;Ac-(iBu)Tyr-Ile-Arg-Leu-Pro-NH₂; Ac-Tyr-Ile-Arg-N(CH₃)O(CH₃);Ac-Tyr-{Ψ(CH₂NH)}-Ile-Arg-Leu-Pro-NH₂; Ac-Tyr-Ile-Arg-NH—CH₂(4-Pyridyl);Ac-Tyr-Ile-{Ψ(CH₂NH)}-Arg-Leu-Pro-NH₂;Ac-Tyr-Chg-Arg(NO₂)-{Ψ(CH₂NH)}-Leu-NH₂;Ac-Tyr-Ile-Arg-{Ψ(COCH₂)}-Gly-Pro-NH₂;Ac-Tyr-Ile-Dab(N^(γ)—C₃H₇N)-Leu-Ala-NH₂; Ac-Tyr-Ile-PalMe(3)-NH₂;Tyr-Ile-Arg-NH₂; D-Tyr-Ile-Arg-Leu-Pro-NH₂,Ac-(Bzl)Gly-(Chx)Gly-(3-guanidopropyl)Gly-NH₂;Cyclo(Gly-Tyr-Ile-Arg-Gly); Tfa-(iBu)Tyr-Chg-Arg-Leu-Pro-NH₂;Ac-pAph-Chg-Arg-Leu-Pro-NH₂; and Ac-Nal(2)-Chg-Arg-Leu-Pro-NH₂.Additional YIR peptides of the invention are shown, for example, inTables 3 and 5.

The present invention also provides a compound having the structureA1—A2—(A3)_(m)—B, wherein R₁ is

R′₁ is selected from the group consisting of H, —CO—R_(a), —SO₂—R_(a),an amino-protecting group, 1 to 6 amino acids, which can be substituted,wherein the N-terminus of said 1 to 6 amino acids is substituted with asubstituent selected from the group consisting of H, —CO—R_(a),—SO₂—R_(a) and an amino-protecting group; and wherein R_(a) is selectedfrom the group consisting of alkyl, aryl and heteroalkyl; R″₁ isselected from the group consisting of H, acyl and alkyl; X is N; R₂ is—CHR₉₉—, wherein R₉₉ is selected from the group consisting of alkyl,aryl, arylalkyl, heteroalkyl and heteroaryl, which can be substitutedwith a substituent selected from the group consisting of 1 to 6 fluoro,chloro, bromo, iodo, amino, nitro, amidino, amido, carboxy, ester, etherand hydroxy groups; R₃ is —C(O)—; R₄ is —NH—; R₅ is —CHR₂₀₁—, whereinR₂₀₁ is an alkyl; R₆ is —C(O)—; R₇ is —NH—; R₈ is —CHR₂₁₀—, wherein R₂₁₀is a heteroalkyl having at least one formal positive charge, wherein theheteroatom is 1 to 6 nitrogen atoms; R₉ is —C(O)—; and B is selectedfrom the group consisting of —OR_(b) and —N—R_(c)R_(d), wherein R_(b) isselected from the group consisting of H, alkyl and a carboxy-protectinggroup, R_(c) is selected from the group consisting of H and alkyl, andR_(d) is selected from the group consisting of alkyl, heteroalkyl and 1to 20 amino acids, which can be substituted with a substituent, whereinthe C-terminus of said compound can be modified with acarboxy-protecting group, a primary amide group or part of a cyclicpeptide as the secondary or tertiary amide group formed with amino groupof R₁. Such a compound can contain one or more amino-protecting groups

For example, a compound of the invention have A1 selected from the groupconsisting of Tyr, F(pNH₂), mAph, pAph and Nal(2), which contain 0 or 1amino-protecting groups; A2 selected from the group consisting of Ileand Chg; A3 selected from the group consisting of Arg, PalMe(3),Dab(N^(γ)—C₃H₇N), Dap(N^(β)—C₃H₇N) and Orn(N^(δ)—C₃H₇N); and B selectedfrom the group consisting of —H, —OH, —NH₂, one to five amino acids orfunctional equivalents thereof and a carboxy-protecting group. Examplesof such compounds include Ac-pAph-Chg-PalMe(3)—NH—CH₂-Chx;Ac-pAph-Chg-PalMe(3)-NH-Chx; Bzf-pAph-Chg-PalMe(3)-NH₂;Ac-pAph-Chg-PalMe(3)-L-P-NH₂; Ac-pAph-Chg-PalMe(3)-NH₂;Cyclopentyl-CO-pAph-Chg-PalMe(3)-NH₂; 3-Iqc-pAph-Chg-PalMe(3)-NH₂;2-Furoyl-pAph-Chg-PalMe(3)-NH₂; 5-Me-thienyl-CO-pAph-Chg-PalMe(3)-NH₂;and Ac-pAph-Chg-PalMe(3)-ol (see, also, Table 5).

The invention further provides a compound having the structure A1—A2—B,i.e., A1—A2—(A3)_(m)—B, wherein m is 0. In such a compound, B can be aheteroarylalkyl such as (4-(N-methyl pyridinium))methyl;2-(3-(N-methylpyridinium))eth-1-yl; 1-(4-(N-methylpyridinium))eth-1-yl;(p-amidino)benzyl; 2-(4-(N-methylpyridinium))prop-2-yl; and2-(4-(N-methylpyridinium))eth-1-yl. Ac-pAph-Chg-AMP(4) andAc-pAph-Chg-AEMP(4) are examples of such compounds.

The invention further provides a non-naturally occurring compound thatspecifically inhibits the activity of factor Xa, having the generalformula A1—A2—(A3)_(m)—B, wherein m is 0 or 1;

wherein A1 is R₁—R₂—R₃; A2 is R₄—R₅—R₆; A3 is R₇—R₈—R₉;

wherein R₁ is selected from the group consisting of:

i) 1 to 20 amino acids;

wherein X is selected from the group consisting of N, CH and NC═O, and

wherein R′₁ and R″₁ independently are selected from the group consistingof —H, alkyl, acyl, aryl, arylalkyl, an amino-protecting group and 1-20amino acids, and

wherein R₁ can be substituted by a substituent;

R₂ is —CR₉₉R₁₀₀—, wherein R₉₉ and R₁₀₀ independently are selected fromthe group consisting of an —H, alkyl, arylalkyl, heteroarylalkyl andheteroaryl, and wherein R₉₉ and R₁₀₀ independently can be substitutedwith a substituent;

R₃ is selected from the group consisting of —C(O)—, —CH₂—, —CHR₉₉—C(O)—and —C(O)—NR₃₅—CH₂—C(O)—, wherein R₃₅ is the CHR₅₅ group of the bridginggroup —C(O)—CR₅₅—;

R₄ is selected from the group consisting of —CH₂— and —NR₅₀—, whereinR₅₀ is selected from the group consisting of H, alkyl, arylalkyl andheterocyclic;

R₅ is —CR₂₀₁R₂₀₂—, wherein R₂₀₁ and R₂₀₂ independently are selected fromthe group consisting of H, alkyl, aryl and arylalkyl, and wherein R₂₀₁and R₂₀₂ independently can be substituted with a substituent;

R₆ is selected from the group consisting of —C(O)—, —CH₂— and—CHR₉₉—C(O)—;

R₇ is selected from the group consisting of —CH₂— and —NR₅₁—, whereinR₅₁ is H, alkyl, arylalkyl, heteroalkyl and heteroarylalkyl, and any ofthese moieties substituted by a substituent selected from the groupconsisting of Q and —(CH₂)_(n)-Q, wherein n is 1 to 5 and wherein Q isselected from the group consisting of an amino, amidino, imidazole andguanidino group, which can be substituted with a substituent, and amono-, di-, tri- or tetra-alkylammonium of a pharmaceutically acceptablesalt, isoureide or isothioureide thereof;

R₈ is —CR₂₁₀R₂₁₁—, wherein R₂₁₀ and R₂₁₁ independently are selected fromthe group consisting of H, alkyl, alkylaryl and heterocyclic, and any ofthese moieties substituted by a substituent selected from the groupconsisting of Q and —(CH₂)_(n)-Q, wherein n is 1 to 5 and wherein Q isselected from the group consisting of amino, amidino, imidazole andguanidino group, which can be substituted with a substituent, and amono-, di-, tri- or tetra-alkylammonium of a pharmaceutically acceptablesalt, isoureide or isothioureide thereof;

R₉ is selected from the group consisting of —C(O)—, —CH₂— and—CHR₉₉—C(O)—; and

wherein, when m is 1, B is selected from the group consisting of 1 to 20amino acids, —NHR₅₂, —NR₆₀R₆₁, —OR₇₀ and —CHR₆₀R₆₁,

wherein R₅₂ is selected from the group consisting of H, alkyl,arylalkyl, heteroarylalkyl and heteroaryl;

wherein R₆₀ and R₆₁ independently are selected from the group consistingof H, alkyl, arylalkyl, aryl, heteroarylalkyl and heteroaryl, and

wherein R₇₀ is selected from the group consisting of H, acyl, alkyl,arylalkyl and heteroarylalkyl,

and wherein when m is 0, B is selected from the group consisting of 1 to20 amino acids, —OR₇₀, —NHR₅₂ and —NR₆₀R₆₁, which is joined to R₆ by anamide bond or an ester bond;

wherein B can be substituted with a substituent, provided that

when R₃ is —CH₂— or —CHR₉₉—C(O)—, R₄ is NR₅₀;

when R₄ is —CH₂—, R₃ is —C(O)— or —CHR₉₉—C(O)—;

when R₆ is —CH₂—, R₇ is —NHR₅₁—;

when R₇ is CH₂, R₆ is —C(O)— or —CHR₉₉—C(O)—;

when R₄ is —NR₅₀— and R₁ is

R₅₀ and R′₁ are taken together to form a bridging group having theformula: —C(O)—CHR₅₅—,

wherein CHR₅₅ represents R₅₀ and the carbonyl group represents R′₁, andR″₁ and R₅₅ independently are H, C₁ to C₆ alkyl or arylalkyl; and whenR₃ is —C(O)—NR₃₅—CH₂—C(O)—, then R₄ is —NR₅₀—, R₁ is

R₃₅ and R′₁ are taken together to form a bridging group having theformula —C(O)CHR₅₅—,

wherein C(O) represents R′₁ and CHR₅₅ represents R₃₅; R″₁ and R₅₅independently are H or a C₁ to C₆ alkyl; further wherein the abovecompound is not one of the following compounds:

a) RYIRF-NH₂(SEQ ID NO: 12);

GNFFRF-NH₂(SEQ ID NO: 13);

KNEFIRF-NH₂(SEQ ID NO: 14);

KHEYLRF-NH₂(SEQ ID NO: 15);

SDPNFLRF-NH₂(SEQ ID NO: 16);

FMRF-NH₂(SEQ ID NO: 17);

FLRF-NH₂(SEQ ID NO: 18);

YMRF-NH₂(SEQ ID NO: 19);

YLRF-NH₂(SEQ ID NO: 20);

pQDPFLRF-NH₂;

SDPFLRF-NH₂(SEQ ID NO: 21);

NDPFLRF-NH₂(SEQ ID NO: 22);

GDPFLRF-NH₂(SEQ ID NO: 23);

SDPYLRF-NH₂(SEQ ID NO: 24);

SDPYFFRF-NH₂(SEQ ID NO: 25);

ALAGDHFFRF-NH₂(SEQ ID NO: 26);

pQDVDHVFLRF-NH₂;

pQDVVHSFLRF-NH₂;

SDRNFLRF-NH₂(SEQ ID NO: 27);

TNRNFLRF-NH₂(SEQ ID NO: 28);

b) H-D-Phe-Phe-Arg-NH-heptyl;

H-D-Phe-Phe-Arg-NH-lauryl;

H-D-Phe-Phe-Arg-NH—OH;

H-D-Phe-Phe-Arg-NH-isopropyl;

H-D-Phe-Phe-Arg-NH₂;

c) H-Phe-Val-Arg-OMe;

H-D-Phe-Val-Arg-H;

d) (Npys)-Cys-Val-Asn-Tyr-Ile-Arg-Lys-Arg-Ser-Leu-Gln-Thr-Val-OH(SEQ IDNO: 29);

(Cys)-Val-Asn-Tyr-Ile-Arg-Lys-Arg-Ser-Leu-Gln-Thr-Val-OH(SEQ ID NO: 30);

e) Asn-Arg-Val-Tyr-Ala-His-Pro-Phe(SEQ ID NO: 31);

Asn-Arg-Val-Tyr-Abu-His-Pro-Phe(SEQ ID NO: 32);

Asn-Arg-Val-Tyr-Nle-His-Pro-Phe(SEQ ID NO: 33);

Asn-Arg-Val-Tyr-aIle-His-Pro-Phe(SEQ ID NO: 34);

Asn-Arg-Val-Tyr-Aev-His-Pro-Phe(SEQ ID NO: 35);

Asn-Arg-Val-Tyr-Cpg-His-Pro-Phe(SEQ ID NO: 36);

Asn-Arg-Val-Tyr-Chg-His-Pro-Phe(SEQ ID NO: 37);

f) compounds of the formula:

X_(F)-Arg-Val-Tyr-Y_(F)-His-Pro-W_(F)(SEQ ID NO: 38) (II)

wherein in the above Formula (II):

 X_(F) stands for sarcosyl, lactoyl or hydroxyacetyl radical;

 Y_(F) is cyclopentylglycyl or cyclohexylglycyl;

 W_(F) is an aliphatic amino acid radical or lactic acid radical;

g) compounds of the formulas:

Z_(G)-X_(G)-Arg(A)-Val-Tyr(B_(G))-Y_(G)-His(E_(G))-Pro-W_(G)-OG (III)and

Z_(G)-X_(G)-Arg(A_(G))-Val-Tyr(B_(G))-Y_(G)-His-Pro-W_(G)-OG (IV);

wherein in the above Formulas (III) and (IV):

Y_(G) is cyclopentylglycyl or cyclohexylglycyl;

W_(G) is an aliphatic amino acid radical or lactic acid radical;

Z_(G) is a protecting group removable by acidolysis or catalytichydrogenation, preferably benzyloxycarbonyl or tert-butoxycarbonyl,

A_(G) is a group suitable for the temporary protection of the guanidinogroup of arginine, preferably a nitro group,

B_(G) is a group suitable for the temporary protection of the aromatichydroxyl group of tyrosine, preferably benzyl or substitute benzyl,

E_(G) is a group suitable for the temporary protection of the imidazolegroup of histidine, preferably dinitrophenyl,

C_(G) is a group suitable for the temporary protection of the C-terminalcarboxyl group, resistant to acid treatment but removable for example bycatalytic hydrogenation, for example benzyl or substituted benzyl, and

X_(G) depending on the meaning of X, represents either a sarcosyl groupor an aliphatic carboxylic acid radical containing an aminooxy group inthe o-position;

and further wherein B_(G) in the above formula is not a chromogenicgroup which is removable by enzymatic hydrolysis and capable of forminga colored or fluorescent compound containing the chromogenic groupB_(G).

The present invention also includes a family of non-naturally occurringcompound that specifically inhibits the activity of factor Xa, havingthe general formula A₁—A₂—(A3)_(m)—B, wherein m is 1 or 0;

wherein A₁ is R₁—R₂—R₃; A₂ is R₄—R₅—R₆; A₃ is R₇—R₈—R₉;

wherein

R₁ is

X is N;

R′₁ is selected from isobutyl, 2-methylpentyl, cyclohexylmethyl,cyclohexenylmethyl, —H, 2-methylbutyl and 2,3-dimethylpentyl;

R″₁ is selected from 2-benzofuroyl, alloc, acetyl, trifluoroacetyl,2-quinolinoyl, 3-pyridoyl, 4-isoquinolinoyl, 5-benzylimidazoyl,2-naphthylmethyl, 5-pyridiminoyl, benzoyl, 2-pyridoyl, tosyl,3-quinolinoyl, 2-naphthylsulfonyl, 2-methylbenzyl, 2-furoyl,3,4-dichlorobenzoyl, 2-thienylacetyl, N(5-methyl-2-thienyl),ethoxycarbonylacetyl, 2-fluorobenzoyl, t-butoxycarbonyl, benzyl and 1-20amino acids;

R₂ is —CR_(2A)R_(2B), wherein —R_(2A) and —R_(2B) are independentlyselected from the group consisting of —H, 4-amidinophenylmethyl,4-aminophenylmethyl, 4-hydroxyphenylmethyl, 2-naphthylmethyl,4-(N-methylpyridinyl)methyl, (3-iodo-4-aminophenyl)methyl,(4-aminocarbonylphenyl)methyl, (3-iodo-4-hydroxyphenyl)methyl,(4-cyanophenyl)methyl, (4-hydroxyphenyl)methyl;

R₃ is —C(O)—;

R₄ is —NH—;

R₅ is —CR_(5A)R_(5B), wherein —R_(5A) and —R_(5B) are independentlyselected from the group consisting of —H, 1-methylpropyl,cyclohexylmethyl;

R₆ is —C(O)—;

R₇ is —NH—;

R₈ is —CR_(8A)R_(8B), wherein —R_(8A) and —R_(8B) are independentlyselected from the group consisting of —H, 3-guanylpropyl,(dimethylamidinium)aminomethyl, (dimethylamidinium)aminoethyl,3-(N-methylpyridinyl)methyl, 4-(N-methylpyridinyl)methyl;

R₉ is —C(O)—; and

B is Leu-Pro-NH₂, Leu-Hyp-NH₂, Pen(CH₂COOH)-Pro-NH₂,Cys(CH₂COOH)-Pro-NH₂, γ-carboxyglutamic acid-Pro-NH₂,(N-carboxymethyl)Gly-Pro-NH₂, (N-carboxyethyl)Gly-Pro-NH₂,(N-1,3-dicarboxypropyl)Gly-Pro-NH₂, (N-methyl)Leu-Pro-NH₂, Leu-NH₂,Leu-OH, —NH-(4-trimethylammoniumbenzyl),—NH-[4-(1-methylpyridinium)methyl], and —NH-(4-amidinobenzyl).

The present invention further includes a sub-family of non-naturallyoccurring compound that specifically inhibits the activity of factor Xa,having the general formula A₁—A₂—(A₃)_(m)—B, wherein m is 1;

wherein A₁ is R₁—R₂—R₃; A₂ is R₄—R₅—R₆; A₃ is R₇—R₈—R₉;

wherein

R₁ is

X is N;

R′₁ is selected from H, isobutyl, 2-methylpentyl, cyclohexylmethyl,3-quinolinyl, 2-methylbutyl, 2,3 dimethyl pentyl, andcyclohexenylmethyl;

R″₁ is selected from 2-benzofuroyl, alloc, acetyl, trifluoroacetyl,2-quinolinoyl, 3-pyridoyl, 4-isoquinolinoyl, 5-benzylimidazoyl,2-naphthylmethylene, 5-pyrazinoyl, benzoyl, 2-pyridoyl, tosyl,3-quinolinoyl, 2-naphthylsulfonyl, 2-methylbenzyl, and benzyl;

R₂ is —CR_(2A)R_(2B), wherein —R_(2A) and —R_(2B) are independentlyselected from the group consisting of —H, 3-amidinophenylmethyl,4-amidinophenylmethyl, 4-aminophenylmethyl, 4-hydroxyphenylmethyl,2-naphthylmethyl, 4-(N-methylpyridinyl)methyl,(3-iodo-4-aminophenyl)methyl, (4-aminocarbonylphenyl)methyl,(3-iodo-4-hydroxyphenyl)methyl, (4-cyanophenyl)methyl, and3-indolylmethyl;

R₃ is selected from the group consisting of —C(O)—, —CH₂—, —CHR₉₉—C(O)—and —C(O)—NR₃₅—CH₂—C(O)—, wherein R₃₅ is the CHR₅₅ group of the bridginggroup —C(O)—CR₅₅—;

R₄ is —NH—;

R₅ is —CR_(5A)R_(5B), wherein —R_(5A) and —R_(5B) are independentlyselected from the group consisting of —H, 2-butyl, cyclohexyl andphenyl;

R₆ is —C(O)—;

R₇ is —NH—;

R₈ is —CR_(8A)R_(8B), wherein —R_(8A) and —R_(8B) are independentlyselected from the group consisting of —H, 3-guanylpropyl,(dimethylamidinium)aminomethyl, (dimethylamidinium)aminoethyl,3-(N-methylpyridinyl)methyl, N(carboxymethyl)(3-pyridinylmethyl), and4-(N-methylpyridinyl)methyl;

R₉ is selected from the group consisting of —C(O)—, —CH₂— and—CHR₉₉—C(O)—; and

B is —NH₂, —OH, Leu-Pro-NH₂, Leu-Hyp-NH₂, Pen(CH₂COOH)-Pro-NH₂,Cys(CH₂COOH)-Pro-NH₂, γ-carboxyglutamic acid-Pro-NH₂,(N-carboxymethyl)Gly-Pro-NH₂, (N-carboxyethyl)Gly-Pro-NH₂,(N-1,3-dicarboxypropyl)Gly-Pro-NH₂, (N-methyl)Leu-Pro-NH₂, Leu-NH₂, andLeu-OH.

Specific embodiments of this sub-family of compounds are taught inExample XXXIX in Table 6 (Compounds 1-86).

The present invention further includes a sub-family of non-naturallyoccurring compound that specifically inhibits the activity of factor Xa,having the general formula A1—A2—(A3)_(m)—B, wherein m is 0;

wherein A₁ is R₁—R₂—R₃; and A2 is R₄—R₅—R₆;

wherein

R₁ is

X is N;

R′₁ is selected from the group consisting of H, alkyl, acyl, aryl,arylalkyl and an amino-protecting group;

R″₁ is selected from 2-furoyl, 3,4-dichlorobenzoyl, 2-thienylacetyl,5-methyl-2-thienoyl, acetyl, ethoxycarbonylacetyl, 2-fluorobenzoyl,alloc, and t-butoxycarbonyl;

R₂ is —CR_(2A)R_(2B)—, wherein R_(2A) and R_(2B) are independentlyselected from the group consisting of an H; alkyl, arylalkyl,heteroarylalkyl and heteroaryl, and wherein R_(2A) and R_(2B)independently can be substituted with a substituent;

R₃ is selected from the group consisting of —C(O)—, —CH₂—, —CHR₉₉—C(O)—and —C(O)—NR₃₅—CH₂—C(O)—, wherein R₃₅ is the CHR₅₅ group of the bridginggroup —C(O)—CR₅₅—;

R₄ is —NH—;

R₅ is —CR_(5A)R_(5B), wherein —R_(5A) and —R_(5B) are independentlyselected from the group consisting of —H, and cyclohexyl;

R₆ is —C(O)—;

B is —NH-(4-trimethylammoniumbenzyl),—NH-[4-(1-methylpyridinium)methyl], —NH-[4-(1-ethylpyridinium)methyl],and —NH-(4-amidinobenzyl).

Specific embodiments of this sub-family of compounds are taught inExample XXXIX in Table 6 (Compounds 87-103).

The choice of including an L- or a D-amino acid in a compound of thepresent invention can depend, in part, on the desired characteristics ofthe peptide. For example, the incorporation of one or more D-amino acidscan confer increased stability on the compound in vitro or in vivo. Theincorporation of one or more D-amino acids also can increase or decreasethe pharmacological activity of the compound. In some cases it can bedesirable to allow the compound to remain active for only a short periodof time. In such cases, the incorporation of one or more L-amino acidsin the compound can allow endogenous peptidases in an individual todigest the compound in vivo, thereby limiting the individual's exposureto the active compound. The skilled artisan can determine the desirablecharacteristics required of compound of the invention by taking intoconsideration, for example, the age and general health of an individual.

A compound of the invention can be chemically synthesized using, forexample, an automated synthesizer (see Example I). Selectivemodification of a reactive group such as a group present on an aminoacid side chain or an N-terminus or a C-terminus reactive group in apeptide can impart desirable characteristics such as increasedsolubility or enhanced inhibitory function to a compound of theinvention.

Where solid phase synthesis methods are employed, the chemicalcomposition of a compound can be manipulated while the nascent peptideis attached to the resin or after the peptide has been cleaved from theresin to obtain, for example, an N-terminal derivative such as anN-terminus acetylated compound. Similar modifications also can be madeto a carboxy group of a compound, including a C-terminus carboxy group,which, for example, can be amidated. One skilled in the art also cansynthesize a YIR peptide of the invention using solution phase organicchemistry. A synthesized compound can be purified using well knownmethods such as reverse phase-high performance liquid chromatography(RP-HPLC; see Example I) or other methods of separation based, forexample, on the size, charge or hydrophobicity of the compound.Similarly, well known methods such as amino acid sequence analysis ormass spectrometry (MS) can be used for characterizing the structure of acompound of the invention (see Example I).

The YIR peptides of the invention can be linear or cyclic (see, forexample, Table 3, below). Cyclization can be accomplished by forming abridge between two nonadjacent residues, moieties or substituents, whichcan be within or outside of the YIR motif. Cyclization also can beaccomplished, for example, by forming a bridge between one of theresidues within the YIR motif and a nonadjacent residue, moiety orsubstituent outside the YIR sequence. For example, peptides orpeptidomimetics can by cyclized via S—S, —CH₂—S—, —CH₂—O—CH₂—, lactam orester linkages or as previously reported (see Hruby, Life Sci.31:189-199 (1982); Toniolo, Int. J. Pept. Prot. Res. 35:287-300 (1990);Kates et al., Tetr. Lett. 34:1549-1552 (1993), each of which isincorporated herein by reference).

As used herein, the phrase “outside the YIR motif” means not including atyrosine, isoleucine or arginine residue of the YIR sequence or itsequivalent present in a YIR peptide of the invention. In contrast, thephrase “within the YIR motif” means including at least one of thetyrosine, isoleucine and arginine residues of the YIR sequence or itsequivalent. The term “bridge” in referring to a cyclic compound means abond formed between two non-adjacent amino acids present in a YIRpeptide of the invention.

Cyclization can be achieved by the formation, for example, of adisulfide bond or a lactam bond between X₁ and X₂. Residues capable offorming a disulfide bond include, for example, Cys, Pen, Mpr, and Mppand its 2-amino group-containing equivalents. Residues capable offorming a lactam bridge include, for example, Asp, Glu, Lys, Orn,α,β-diaminopropionic acid, α-amino-adipic acid, α,γ-diaminobutyric acid,diaminoacetic acid, aminobenzoic acid and mercaptobenzoic acid. Thecompounds disclosed herein can be cyclized, for example, via a lactambond, which can utilize a side chain group of one non-adjacent residueto form a covalent attachment to the N-terminus amino group of X₁ or ofY. Alternative bridge structures also can be used to cyclize thecompounds of the invention, including, for example, peptides andpeptidomimetics, which can be cyclized via S—S, —CH₂—S—, —CH₂—O—CH₂—,lactam, ester or other linkages (see for example, Hruby, supra, 1982;Toniolo, supra, 1990; Kates et al., supra, 1993).

A composition of the present invention can be provided as a homogenouscomposition or as a mixture of compounds containing various combinationsof substituents. The flexibility permitted by the choice of substituentspermits a great deal of control over the physico-chemical properties ofthe peptide compound analogs. The choice of the substituent alsoinfluences the binding affinity of the compound (see Examples).

Various compounds containing different arrangements of the substituentsexhibit different levels of inhibitory activity for factor Xa. Thesecompounds were synthesized according to the procedures described in theExamples. Testing the peptides for inhibitory activity was accomplishedusing the assays described in Examples XXXVII and XXXVIII. Using suchmethods, one skilled in the art can synthesize a compound as disclosedherein, including a modification thereof, and determine the factor Xainhibitory activity of the compound.

The invention provides compounds that specifically inhibit factor Xaactivity. Such compounds have a K_(i)≦100 μM, preferably ≦2 nM, forfactor Xa activity and do not substantially inhibit the activity ofother proteases involved in coagulation and fibrinolysis relative to theinhibition of factor Xa (see Table 2, above). Such other proteasesinclude, for example, thrombin and plasmin. Specificity of the compoundsof the invention is demonstrated in Example XXXVII, below (see, also,Table 2, above).

A compound of the invention can be used advantageously as ananticoagulant, which can be contacted with a blood sample to preventcoagulation. For example, an effective amount of a compound of theinvention can be contacted with a freshly drawn blood sample to preventcoagulation of the blood sample. As used herein, the term “effectiveamount” when used in reference to a compound of the invention means anamount of a compound that inhibits factor Xa activity. The skilledartisan would recognize that an effective amount of a compound of theinvention can be determined using the methods disclosed herein (seeExamples XXXVII and XXXVIII) or otherwise known in the art. In view ofthe disclosed utility of a compound of the invention, the skilledartisan also would recognize that an agent such as heparin can bereplaced with a compound of the invention. Such a use of a compound ofthe invention can result, for example, in a cost saving as compared toother anticoagulants.

In addition, a compound of the invention can be administered to anindividual for the treatment of a variety of clinical conditions,including, for example, the treatment of a cardiovascular disorder or acomplication associated, for example, with infection or surgery.Examples of cardiovascular disorders include restenosis followingangioplasty, adult respiratory distress syndrome, multi-organ failure,stroke and disseminated intravascular coagulation clotting disorder.Examples of related complications associated with surgery include, forexample, deep vein and proximal vein thrombosis, which can occurfollowing surgery. Thus, a compound of the invention is useful as amedicament for reducing or inhibiting unwanted coagulation in anindividual.

Since a YIR peptide of the invention can inhibit factor Xa activity,such a compound can be useful for reducing or inhibiting blood clottingin an individual. As used herein, the term “individual” means avertebrate, including a mammal such as a human, in which factor Xa isinvolved in the clotting cascade.

Blood clotting in an individual can be reduced or inhibited byadministering to the individual a therapeutically effective amount of aYIR peptide of the invention. As used herein, the term “therapeuticallyeffective amount” means the dose of a YIR peptide that must beadministered to an individual in order to inhibit factor Xa activity inthe individual. More specifically, a therapeutically effective amount ofa compound of the invention inhibits factor Xa catalytic activity eitherdirectly, within the prothrombinase complex or as a soluble subunit, orindirectly, by inhibiting the assembly of factor Xa into theprothrombinase complex. In particular, such compounds can inhibit factorXa activity with a K_(i)≦100 μM and, preferably, with a K_(i)≦2 nM. Atherapeutically effective amount can be determined using the methodsdescribed, for example, in Examples XXXVII and XXXVIII or otherwiseknown in the art.

In the practice of a therapeutic method of the invention, the particulardosage to obtain a therapeutically effective amount of a pharmaceuticalcomposition to be administered to the individual will depend on avariety of considerations, including, for example, the nature orseverity of the disease, the schedule of administration and the age andphysical characteristics of the individual. An appropriate dosage can beestablished using clinical approaches well known in the medical art.Thus, the invention provides a method of specifically inhibiting factorXa activity by contacting factor Xa with a compound having the sequenceX₁-YIR-X₂ or A1—A2—(A3)_(m)—B, where m is 0 or 1, or a functionalequivalent thereof. The invention further provides a method of reducingor inhibiting the formation of a blood clot in an individual byadministering a therapeutically effective amount of a compound of theinvention.

A compound of the invention generally will be administered to anindividual as a composition containing the compound and apharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable carrier” refers to a medium or composition that is non-toxicto an individual or has acceptable toxicity as determined by theappropriate regulatory agency. As used herein, the term pharmaceuticallyacceptable carrier encompasses any of the standard pharmaceuticalcarriers such as phosphate buffered saline, water, an emulsion such asan oil/water or water/oil emulsion, or any of various types of wettingagents. Suitable pharmaceutical carriers and their formulations aredescribed by Martin (in Remington's Pharmaceutical Sciences, 15th Ed.(Mack Publishing Co., Easton 1975) which is incorporated herein byreference). Such compositions will, in general, contain atherapeutically effective amount of a compound of the invention togetherwith a suitable amount of carrier so as to comprise the proper dosagefor administration to an individual. Thus, the claimed compounds can beuseful as medicaments for inhibiting factor Xa activity and bloodclotting in an individual.

Pharmaceutically acceptable carriers also can include, for example,other mediums, compounds or modifications to a factor Xa inhibitorcompound that enhances its pharmacological function. A pharmaceuticallyacceptable medium can include, for example, an acid addition salt suchas a salt formed with an inorganic acid such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid or perchloric acid, orwith an organic acid such as acetic acid, oxalic acid, maleic acid,malic acid, tartaric acid, citric acid, succinic acid or malonic acid.Other pharmaceutically acceptable salts include, for example, inorganicnitrate, sulfate, acetate, malate, formate, lactate, tartrate,succinate, citrate, p-toluenesulfonate, and the like, including, but notlimited to, cations based on the alkali and alkaline earth metals suchas sodium, lithium, potassium, calcium or magnesium, as well asnon-toxic ammonium, quaternary ammonium and amine cations such asammonium, methylammonium, dimethylammonium, trimethylammonium,tetramethylammonium, ethylammonium, triethylammonium andtetraethylammonium.

Examples of modifications that enhance the pharmacological function ofthe compound include, for example, esterification such as the formationof C₁ to C₆ alkyl esters, preferably C₁ to C₄ alkyl esters, wherein thealkyl group is a straight or branched chain. Other acceptable estersinclude, for example, C₅ to C₇ cycloalkyl esters and arylalkyl esterssuch as benzyl esters. Such esters can be prepared from the compoundsdescribed herein using conventional methods well known in the art ofpeptide chemistry.

Pharmaceutically acceptable modifications also can include, for example,the formation of peptide amides. Such amide modifications, which can beeffected upon the compounds of the invention, include, for example,those derived from ammonia, primary C₁ to C₆ dialkyl amines, where thealkyl groups are straight or branched chain, or arylamines havingvarious substitutions. In the case of secondary amines, the amine alsocan be in the form of a 5 or 6 membered heterocycle containing, forexample, a nitrogen atom. Methods for preparing such amides are wellknown in the art.

In another embodiment of the invention, a YIR peptide can be used in anassay to identify the presence of factor Xa or to isolate factor Xa in asubstantially purified form. Preferably, the compound of the inventionis labeled with, for example, a radioisotope, and the labeled compoundis detected using a routine method useful for detecting the particularlabel. In addition, a YIR peptide can be used advantageously as a probeto detect the location or amount of factor Xa activity in vivo, in vitroor ex vivo.

It is understood that modifications that do not substantially affect theactivity of the various embodiments of this invention are includedwithin the invention disclosed herein. Accordingly, the followingexamples are intended to illustrate but not limit the present invention.

EXAMPLE I Peptide Synthesis Procedures

Starting materials used in the synthesis were obtained from chemicalvendors such as Aldrich, Sigma, Fluka, Nova Biochem and AdvanceChemtech. During the synthesis of these compounds, the functional groupsof the amino acid derivatives used in these methods were protected byblocking groups to prevent side reaction during the coupling steps.Examples of suitable protecting groups and their use are described inThe Peptides, supra, 1981, and in Vol. 9, Udenfriend and Meienhofer ed.1987, which is incorporated herein by reference.

General solid-phase peptide synthesis was used to produce the compoundsof the invention. Such methods are described, for example, by Stewardand Young (Solid Phase Peptide Synthesis (Freeman and Co., SanFrancisco, 1969), which is incorporated herein by reference).

Unless indicated otherwise, peptides were synthesized on polystyreneresin cross-linked with 1% divinylbenzene. An acid sensitive linker(Rink Linker) was coupled to the solid support (Rink, Tetr. Lett.28:3787 (1987); Sieber, Tetr. Lett. 28:2107 (1987), each of which isincorporated herein by reference). Coupling was performed usingN,N′-diisopropylcarbodiimide (DIC) in the presence of an equivalentamount of HOBt. All couplings were done in either N,N-dimethylformamide(DMF) or DMF:dichloromethane (1:1 mixture) at room temperature (RT) for40 min. Completion of coupling was monitored by ninhydrin test.

Deprotection of the Fmoc group was accomplished using 50% piperidine inDMF for 10 min. The amount of Fmoc released was determined from theabsorbance at 300 nm of the solution after deprotection, volume ofwashes and weight of the resin used in the synthesis. A second (double)coupling was performed where coupling in the first instance wasincomplete. The cycle of each coupling and methods was as follows:

Step Action Reagent and Solvent 1. 1 g Peptide Resin 10 ml DMF 2. 2.4fold-excess amino acid derivative 3. 2.4 equivalent DIC 4. 2.4equivalent HOBt 5. Couple for 40 min 6. Wash (3 × 8 ml) DMF 7. Ninhydrintest 8. Deprotection (10 min) 8 ml 50% Piperidine/DMF 9. Wash (6 × 8 ml)DMF 10. Wash (2 × 8 ml) Dichloromethane (DCM) 11. Ninhydrin test 12.Repeat starting at step 2.

After completion of peptide assembly on the resin, the final Fmocdeprotection was performed, then followed by normal wash cycles anddetermination the amount of Fmoc group released by deprotection. In somecases, the N^(α)-unprotected peptide was acetylated by shaking thepeptide resin with 20-fold excess of acetic anhydride/pyridine (1:1) inDCM for 15 min. The peptide resin was washed successively with DCM, DMFand DCM, then dried under vacuum.

Peptide resin was suspended in reagent K (King et al., Int. J. Pept.Prot. Res. 36:255-266 (1990), which is incorporated herein by reference)cocktail (5 ml/g peptide resin) for 180 min at RT, then the cleavagemixture was filtered in anhydrous diethyl ether and the solidprecipitate was isolated by centrifugation and dried in vacuum oversolid pellets of KOH. The dried peptide was subjected to HPLCpurification using an appropriate gradient of 0.1% TFA in water andacetonitrile (ACN). After collecting the peak containing the intendedsynthetic product, the peptide solution was lyophilized and the peptidewas subjected to an identification process, which included electrosprayMS and amino acid analysis to confirm that the correct compound wassynthesized.

For peptide purification, a sample of crude lyophilized peptide wasdissolved in a mixture of 0.1% aqueous TFA containing 10% to 50% ACN.The peptide solution usually was filtered through a syringe connected toa 0.45 μm nylon “ACRODISC” 13 (Gelman Sciences; Ann Arbor Mich.) filter.A proper volume of filtered peptide solution was injected into asemi-preparative C18 column (Vydac Protein and Peptide C18, 218TP1010;The Separation Group; Hesperia Calif.). The flow rate of a gradient orisocratic mixture of 0.1% TFA buffer and ACN (HPLC grade) as an eluentwas maintained using a Beckman “SYSTEM GOLD” HPLC. Elution of thepeptide was monitored by UV detection at 230 nm (Beckman, System Gold,Programmable Solvent Module 126 and Programmable Detector Module 166controlled by “SYSTEM GOLD” software). After identifying the peakcorresponding to the compound under synthesis, using MS, the compoundwas collected, lyophilized and biologically tested. MS was performedusing a SCIEX API III+ instrument. In addition, NMR was performed usinga General Electric instrument (300 MHz). For NMR, samples typically weremeasured in hexadeuterodimethylsulfoxide or deuterochloroform (CDCl₃;Aldrich).

Amino acid aldehydes were prepared using methods well known in the art.Amino acids and peptide aldehydes have been reported, for example, byFehrentz and Castro, Synthesis 676 (1983); Bajusz et al., J. Med. Chem.33:1729 (1990); Kawamura et al., Chem. Pharm. Bull. 17:1902 (1969), andSomeno et al., Chem. Pharm. Bull., 34:1748 (1986), each of which isincorporated herein by reference. Synthesis of reduced peptide bonds wasperformed at the level of the dipeptide in solution (e.g.,Tyr-{Ψ(CH₂NH)}-Ile), then the properly protected dipeptide was coupledto the rest of the peptide on resin using solid phase peptide synthesis.Alternatively, the protected amino acid aldehyde was coupled to thepeptide on resin using methods described by Ho et al. (Pept. Res.6:10-12 (1993), and references cited therein, each of which isincorporated herein by reference).

EXAMPLE II Synthesis of Ac-Tyr-Ile-Arg-Leu-Ala-NH₂

For the synthesis of Ac-Tyr-Ile-Arg-Leu-Ala-NH₂, 1 g of Rink resin (0.6mmol NH₂/g resin) was used in the procedure as described above. Theresultant peptide was analyzed by MS. (M+H)⁺ found 659.4, calculated(calc.) 659.9.

EXAMPLE III Synthesis of Ac-Tyr-Ile-Arg-Leu-Pro-NH₂

For the synthesis of Ac-Tyr-Ile-Arg-Leu-Pro-NH₂, 1 g of Rink resin (0.6mmol NH₂/g resin) was used in the procedure as described in Example I.The resultant peptide had an (M+H)⁺ found 685.4, calc. 685.9.

EXAMPLE IV Synthesis of Ac-(iBu)Tyr-Ile-Arg-Leu-Pro-NH₂

1 g of Rink resin (0.6 mmol NH₂/g resin) was used. The general solidphase synthesis outlined above was used. After deprotection of Tyr andproper washing of the peptide resin, 50 eq isobutyraldehyde in DMFcontaining 2% glacial acetic acid was added. The resulting mixture wasshaken for 4 hr at RT. After washing the peptide resin with DMFcontaining 2% acetic acid (2×8 ml), 1 g of NaBH₃CN in 10 ml of DMFcontaining 2% acetic acid was added. The peptide resin was shaken for 30min, then the peptide resin was filtered and a fresh mixture of NaBH₃CNin DMF/acetic acid was added and the reaction continued for anadditional 30 min.

The peptide resin then was washed with DMF/2% acetic acid (2×8 ml) andDMF (2×8 ml). The resultant monoalkylated peptide resin was acetylatedwith acetic anhydride triethylamine mixture in DMF (30 eq, 6 h). Afterproper washing of the peptide resin, the peptide was cleaved anddeprotected as described in Example I. HPLC purified peptide wasanalyzed by MS. (M+H)⁺ found 758.4, calc. 758.5.

EXAMPLE V Synthesis of Tfa-(iBu)Tyr-Ile-Arg-Leu-Pro-NH₂(SEQ ID NO: 39)

The same protocol as described in Example IV was used to prepare(iBu)Tyr-Ile-Arg-Leu-Pro-Rink resin. Final trifluoroacetylation wasperformed by treating the peptide-resin with 0.7 Mtrifluoroacetanhydride in the presence of Diisopropylethylamine (DIEA)and N-methyl imidazole (NMI) (1:3:0.3 eq) for 45 min. Cleavage of thepeptide from the resin and isolation of the peptide were performed asdescribed in Example IV. The purified peptide was identified by MS.(M+H)⁺ found 812.4, calc. 812.5.

EXAMPLE VI Synthesis of Ac-Tyr-Ile-Arg-N(CH₃)O(CH₃)

The synthesis of Boc-Arg(N^(G)-Tos)-N(CH₃)O(CH₃) was accomplishedaccording to the literature procedure (Fehrentz and Castro, supra,1983). Boc-Arg(N^(γ)-Tos)-N(CH₃)O(CH₃) (200 mg) was mixed with 5 mltrifluoroacetic acid (TFA) at RT and stirred for 20 min. Disappearanceof the starting material was monitored by thin layer chromatography(TLC) using CHCl₃:MeOH:CH₃COOH (90:9:1) and visualized by ninhydrinspray and UV illumination. Evaporation of the remaining TFA under vacuumand drying in vacuum over KOH pellets resulted in a solid materialhaving the proper mass. (M+H)⁺ found 371.2, calc. 371.4.

In one flask, 150 mg of the material prepared above was dissolved in 1ml DMF, then 57 μl triethylamine was added and the mixture was cooled to0° C. In a second flask, 171 mg of Z-Tyr-Ile-OH (Biochem BioscienceInc.; Philadelphia Pa.) was dissolved in anhydrous tetrahydrofuran (THF)and cooled to −10° C., then 44 μl NNM and 52 μl isobutylchloroformatewas added under N₂ and the mixture was stirred for 15 min. A solution ofArg(Tos)N(CH₃)OCH₃ in DMF previously prepared was added to the mixedanhydride of Z-Tyr-Ile-OH dipeptide and the mixture was stirred at −10°C. for 30 min, then overnight at RT.

After workup of the reaction mixture as described in Example I, thepeptide was dried under vacuum and a small portion was purified by HPLCand analyzed by MS; the peptide had the expected molecular weight (781).The resulting peptide Z-Tyr-Ile-Arg(Tos)-N(CH₃)OCH₃ was mixed with 500μl anisole and subjected to HF deprotection by the usual procedure.After workup, 169 mg of the product Tyr-Ile-Arg-N(CH₃)C(CH₃) wasisolated and identified by MS (found 493.6, calc. 494). The residualpeptide then was dissolved in 1 ml of the 1 N HCl and lyophilized.

Tyr-Ile-Arg-N(CH₃)OCH₃.2HCl (76 mg) was dissolved in ACN, cooled to 0°C. and 13 μl pyridine was added, followed by 15 μl acetic anhydride. Themixture was stirred at 0° C. for 3 hr and completion of the reaction wasmonitored by the ninhydrin test. After stirring at RT for 8 hr, thereaction mixture was worked up and the product,Ac-Tyr-Ile-Arg-N(CH₃)OCH₃, was characterized by MS (found 535.6, calc.535.3).

EXAMPLE VII Synthesis of Ac-Tyr-{Ψ(CH₂NH)}-Ile-Arg-Leu-Pro-NH₂

a. Synthesis of Fmoc-Tyr(But)-H

4.6 g (10.0 mmol) Fmoc-Tyr(But)-OH, 2.1 g (10.1 mmol)dicylohexylcarbodiimide (DCC), 1.26 g (10.1 mmol) benzylmercaptan and0.12 g DMAP were reacted in DCM as described by Ho and Ngu (J. Org.Chem. 58:2315 (1993), which is incorporated herein by reference). Afterworkup, Fmoc-Tyr(But)-S—CH₂C₆H₅ was isolated and, upon reduction of thethioester by stirring with triethylsilane in the presence of 10% Pd oncarbon and purification by flash chromatography, gave a 81% yield ofFmoc-Tyr(But)-H. The NMR and mass of the product were in accordance withthe expected range.

b. Synthesis of Fmoc-Tyr(But)-{Ψ(CH₂NH)}-Ile-(O-Allyl)

0.73 g (1.66 mmol) Fmoc-Tyr(But)-OH and 0.209 g (3.32 mmol) NaBH₃CN in20 ml of 1% AcOH in DMF were added to a solution of 0.516 g (1.82 mmol)TFA.Ile-(O-Allyl) in 2 ml of DMF. After 2 hr, the reaction mixture wasworked up and the final product purified by flash chromatography (ethylacetate:hexane, 35:65) to give an oil product having the proper NMR andMS. (M+H) found 599, calc. 598.7.

c. Synthesis of Fmoc-Tyr(But)-{Ψ(CH₂NH)}-Ile-OH

To 0.467 g (0.78 mmol) Fmoc-Tyr(But)-{Ψ(CH₂NH)}-Ile-OAllyl in 10 ml DCM,was added 89 μl (1.56 mmol) HOAc, 20 μl triethylamine (TEA) and 0.02 gof complex PdCl₂(Ph₃)₂. 231 μl (0.86 nmol) Bu₃SnH was added in oneportion and the mixture was stirred for 1 hr at RT. After proper workupof the reaction mixture, the product was purified on flashchromatography (CHCl₃:MeOH, 20:1) to give a 69% yield (0.319 g) of theexpected peptide. (M+H⁺) found 559, calc. 558.Fmoc-Tyr(But)-{Ψ(CH₂NH)}-Ile-OH then was coupled toArg(Pmc)-Leu-Pro-Rink resin using general solid phase methodology asoutlined in Example I. The finished peptide resinAc-Tyr(But)-{Ψ(CH₂NH)}-Ile-Arg(Pmc)-Leu-Pro-Rink was deprotected andcleaved as usual as described in Example I and purified by HPLC on C18column.

EXAMPLE VIII Synthesis of Ac-Tyr-Ile-Arg-NH—CH₂(4-Pyridyl)

Oxime resin (DeGrado and Kaiser, J. Org. Chem. 45:1295 (1980) (0.862 gof 0.6 mmol/g) was coupled overnight with Boc-Arg(Tos)-OH in thepresence of DIC/HOBt. The resin was washed with DMF, then DCM andacetylated with acetic anhydride/DIEA (1:1 eq) in DCM. After washing theresin with DCM, DMF and DCM, it was deprotected with 25% TFA in DCM for30 min. The deprotected resin was washed with DCM, isopropanol and DCM.To TFA.Arg(Tos)-OxmR was coupled Boc-Ile-OH in symmetrical anhydrideform (3 eq) in the presence 1.5 eq DIEA in DCM. The cycle of washing,acetylation and deprotection, as described above, was repeated. Afterdeprotection, Boc-Tyr(2-BrZ)-OH was coupled in a similar way as Ile,then the finished peptide resin Boc-Tyr(2-BrZ)-Ile-Arg(Tos)-OxmR wasdeprotected and acetylated to give Ac-Tyr(2-BrZ)-Ile-Arg(Tos)-OxmR. Thepeptide resin was dried under vacuum to give a total gain of 0.216 g.

To ⅓ of the resin was added 100 μl (800 μmol) 4-(dimethylamino)pyridinein the presence of 60 μl glacial acetic acid and 120 μl DIEA in 6 ml ofDCM. The resin was shaken overnight at RT. After filtration of the DCMsolution, the resin was washed with 3 ml DMF and the washes werecombined with the DCM filtrate. After evaporation of the solvent, theresidual peptide was deprotected with HF/anisole and processed as usualto obtain the expected peptide. Electrospray MS was performed. (M+H)⁺found 582.3, calc. 582.

EXAMPLE IX Synthesis of Ac-Tyr-Ile-{Ψ(CH₂NH)}-Arg-Leu-Pro-NH₂

a. Synthesis of Boc-Ile-H

Aldehyde was synthesized from 1 g Boc-Ile-N(Me)OMe as described byFehrentz and Castro (supra, 1983). The aldehyde was identified by TLCand NMR as described in the reference.

b. Synthesis of Arg(Tos)-Leu-Pro-MBHA

Synthesis of tripeptide resin was performed by general solid-phaseapproach described in Example I.

c. Synthesis of Boc-Ile-{Ψ(CH₂NH)}-Arg(Tos)-Leu-Pro-MBHA

Boc-Ile-H was coupled to the tripeptide resin Arg(Tos)-Leu-Pro-MBHA byreductive amination using NaBH₃CN in DMF containing 1% acetic acid. TheBoc-group was cleaved as usual and Ac-Tyr-OH was coupled using DIC/HOBt.The finished peptide resin (0.7 g) was deprotected and cleaved from theresin using HF/thioanisole mixture. 19 mg of the crudeAc-Tyr-Ile-{Ψ(CH₂NH)}-Arg-Leu-Pro-NH₂ was HPLC purified on C18 column togive about 5 mg of >90% pure expected peptide. (M+H⁺) found 688.4, calc.687.9.

EXAMPLE X Synthesis of Ac-Tyr-Ile-Dab(N^(γ)—C₃H₇N)-Leu-Ala-NH₂

0.2 g SCAL-TG (0.2 mmol NH₂/g) (Patek & Lebl, Tetr. Lett. 32:3891-3894(1991), which is incorporated herein by reference) was coupled withFmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Ile-OH andFmoc-Tyr(But)-OH using methods as described in Example I. Afteracetylation of the N-terminus and side chain deprotection by TFA, thepeptide resin (SEQ ID NO: 40) Ac-Tyr-Ile-Dab-Leu-Ala-SCAL-TG was washed,neutralized and treated with 0.3 M PyBroP/NMI in DMF for 2 hr. Thefinished peptide was cleaved from the resin using 1 M triphenylphosphine/(CH₃)₃SiCl in DCM (3×1 hr), followed by 100% TFA (1 hr). Afterisolation of the crude peptide by diethyl ether precipitation, thepeptide was lyophilized from a 0.1% aqueous solution of TFA. The peptideAc-Tyr-Ile-Dab(N^(γ)—C₃H₇N)-Leu-Ala-NH, was purified by HPLC andcharacterized by MS. (M+H⁺) found 676.4, calc. 676.4.

EXAMPLE XI Synthesis of Ac-Tyr-Ile-PalMe(3)-NH₂

To 1.0 g Rink resin (0.48 mmol NH₂/g) was coupled Fmoc-Pal(3)-OH,Fmoc-Ile-OH and Fmoc-Tyr(But)-OH using the methods described in ExampleI. To 0.25 g of the finished peptide resin,Fmoc-Tyr(But)-Ile-Pal(3)-Rink, was added 500 μl methyl iodide (MeI) inDCM and the peptide resin was shaken for 6 hr. The finished peptideresin, Fmoc-Tyr(But)-Ile-PalMe(3)-Rink, was deprotected and acetylatedand cleaved as described in Example I. A portion of the crude peptidewas purified by HPLC and the final peptide was characterized by MS.

EXAMPLE XII Synthesis of Ac-Cyclo(Glu-Tyr-Ile-Arg-Leu-Lys)-NH₂(SEQ IDNO: 41)

1 g SCAL-TG (0.29 mmol NH₂/g) (see Example X) was coupled withFmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Ile-OH,Fmoc-Tyr(But)-OH and Fmoc-Glu(OtBu)-OH using methods as described inExample I. After Fmoc removal, the peptide resin was acetylated andwashed with DMF, then with DCM. The peptide resin,Ac-Glu(OtBu)-Tyr(But)-Ile-Arg(Pmc)-Leu-Lys(Boc)-SCAL-TG, was deprotectedwith reagent K, washed, neutralized and cyclized using BOP/HOBt/DIEA(5:5:5 eq) in DMF for 2 hr. The completion of coupling was monitored byninhydrin test as described by Kaiser (Kaiser et al., Anal. Biochem.34:595 (1970), which is incorporated herein by reference). Aftercyclization, the peptide was cleaved from the resin, purified by HPLCand characterized by MS. (M+H)⁺ found: 844.5, calc. 844.5.

EXAMPLE XIII Synthesis of Cyclo(Gly-Tyr-Ile-Arg-Gly)

1 g oxime resin (see Example VIII) (0.6 mmol NH₂/g) was coupledovernight with Boc-Gly-OH in the presence of DIC/HOBt. After washing anddeprotection of the resin, Boc-Arg(Tos)-OH, Boc-Ile-OH andBoc-Tyr(2-BrZ)-OH were coupled using methods as described in ExampleVIII. One-third of the peptide resin,Boc-Tyr(2-BrZ)-Ile-Arg(Tos)-Gly-Oxime resin, was deprotected and coupledwith Boc-Gly by DIC/HOBt. The finished peptide resin was deprotected,neutralized and cyclized overnight in DMF containing 1% acetic acid. Theresin was filtered and washed (DMF), the filtrates were combined and theorganic solvent was removed by evaporation in vacuo. The residualpeptide was deprotected (HF/anisole), lyophilized, HPLC purified andcharacterized by M.S. (M+H)⁺ found; 547.8, calc. 547.8.

EXAMPLE XIV Synthesis of N-substituted Glycine Compounds

Synthesis of Ac-(Bzl)Gly-(Chx)Gly-(3-guanidopropyl)Gly-NH₂

For the synthesis of N-substituted glycines, the procedure of Zuckermannet al. (J. Am. Chem. Soc. 114:10646 (1992), which is incorporated hereinby reference) was used. 1 g SCAL-TG (0.29 mmol NH₂/g) (see Example X)was coupled with bromoacetic acid via symmetrical anhydride in DCM/DMF.Each coupling reaction was repeated twice. To Br—CH₂CO-SCAL-TG resin wasadded Boc-NH—CH₂CH₂CH₂NH₂ in DMSO and the resin was rocked for 2 hr.After deprotection, the process repeated by alternating the coupling ofBr—CH₂COOH to the resin and the reaction of bromoacetic acid resin withthe proper amine. The (Bzl)Gly-(Chx)Gly-(Boc-NH—(CH₂)₃)Gly-SCAL-TG resinwas acetylated with acetic anhydride/DIEA/NMI (1:1:0.25) in DMFovernight. After deprotection of the Boc group, the resin,Ac-(Bzl)Gly-(Chx)Gly-(3-aminopropyl)Gly-SCAL-TG, was treated with 1.8 Mcarboxyamidinopyrazole.HCl (Bernatowicz et al., J. Org. Chem.57:2497-2502 (1992), which is incorporated herein by reference) in ofpresence of DIEA (1:1) in DMF for 3 h at RT. The completion ofguanylation was monitored by the Kaiser test. Cleavage and processing ofthe resultant peptide was performed as described in Example X andanalyzed by M.S. (M+H)⁺ found 502.3, calc. 502.3.

EXAMPLE XV Synthesis of Diketopiperazine Compounds

Synthesis of Cyclo(Ser-Ida)-Ile-Arg-Leu-Ala-NH₂(SEQ ID NO: 42)

The starting protected tetrapeptide, Fmoc-Ile-Arg(Pmc)-Leu-Ala-Rink, wasprepared by Fmoc strategy on Rink resin (see Example I). After Fmocdeprotection of the peptide resin, Fmoc-Ida(OMe)-OH (3 eq; DIC, HOBt)and Fmoc-Ser(tBu)-OH (7 eq; symmetrical anhydride) were coupledconsecutively. The final deprotection and spontaneous ring closure wereperformed simultaneously by 1 hr exposure to 50% piperidine/DMF. Afterwashing steps, the final peptide was cleaved and deprotected usingTFA/thioanisole/H₂O (95:2.5:2.5). The resultant peptide was processed asdescribed above and it was analyzed by HPLC (>95%) and by MS. (M+H)⁺found 655.4, calc. 655.38.

EXAMPLE XVI Synthesis of Ph-C(NOCH₂Ph)-CO-I-R-NH₂

0.2 g Rink resin was coupled with Fmoc-Arg(Pmc)-OH, Fmoc-Ile-OH,followed by removal of Fmoc protection (see Example I). To the peptideresin, Ile-Arg(Pmc)-Rink, was coupled with Ph-C(NOCH₂Ph)-COOH using theDIC/HOBt protocol described above. The finished peptide resin,Ph-C(NOCH₂Ph)-CO-Ile-Arg(Pmc)-Rink, was worked up as described inExample I and analyzed by MS. (M+H)⁺ found 524.3, calc. 524.6.

EXAMPLE XVII Synthesis of Ac-pAph-Ile-Arg-Leu-Pro-NH₂(SEQ ID NO: 43)

The synthesis was performed on 100 mg Rink resin (0.48 mmol/g) accordingto the method of Example I, using the following amino acids derivatives:Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Arg (Pmc) —OH, Fmoc-Ile-OH andFmoc-pAph-(Fmoc)-OH (racemic mixture). The cleavage and isolation of thepeptide were carried out as described in Example I. Both diastereomericpeptides were isolated by RP-HPLC and identified by MS. (M+H)⁺ found754.4. calc. 754.5.

EXAMPLE XVIII Synthesis of Ac-Tyr-Chg-Arg-ol

The peptide sequence was built on 0.25 g Fmoc-Arg(Pmc)-Sasrin resin (0.5mmol NH₂/g resin; Bachem Bioscience) using the method described inExample I. After N-terminus Fmoc deprotection and acetylation, theprotected peptide was cleaved from the resin by reductive cleavage as aC-terminus alcohol (Mergler et al., Peptides pp. 177-178 (eds. Schneiderand Eberle; Leiden 1993), which is incorporated herein by reference).The peptide resin was shaken with a solution of NaBH₄ (4 eq) in 2 mlTHF:EtOH (6:1) for 24 hr. Following the cleavage reaction, the resin waswashed with DCM, then the cleavage solution and washes were combined andlyophilized. The lyophilized peptide was deprotected by treatment withTFA/water/thioanisole (90:5:5) for 2 hr and isolated by precipitation.HPLC purified peptide was analyzed by MS. (M+H)⁺ found 505.3, calc.505.3.

EXAMPLE XIX Synthesis of Ac-Tyr-Chg-Arg-ol.acetate

The protected peptide alcohol was prepared as described in ExampleXVIII. 10 mg crude material was dissolved in DCM/ACN and treated withacetic anhydride (2 mmol) in the presence of TEA (2.4 mmol) for 20 min.The solution was filtered, evaporated and the peptide was deprotected asdescribed above. HPLC purified peptide was analyzed by MS. (M+H)⁺ found547.3, calc. 547.3.

EXAMPLE XX Synthesis of Ac-Phe(pNH₂)-Chg-Orn(C(NH)CH₃)-Leu-Pro-NH₂

1 g “TENTAGEL S” NH₂ resin (0.28 mmol NH₂/g resin; Rapp Polymer;Tubingen Germany) was functionalized with SCAL linker as described inExample X and the following amino acids were coupled: Fmoc-Pro-OH;Fmoc-Leu-OH; Fmoc-Orn(Boc)-OH and Fmoc-Chg-OH. The peptide resinFmoc-Chg-Orn(Boc)-Leu-Pro-SCAL-TG was treated with 50% TFA in DCM (1wash for 1 min, then 1 wash for 30 min), washed 3× with DCM, neutralizedwith 5% DIEA in DCM (2×30 sec.) and 2× with DCM. To the peptide resinwas added a solution of 1.5 g ethyl acetimidate hydrochloride (Aldrich)in 4 ml 1:1 pyridine:DIEA and 3 ml DMF and the coupling was continuedovernight at RT.

The peptide resin, Fmoc-Chg-Orn(C(NH)CH₃)-Leu-Pro-SCAL-TG, wasdeprotected with 20% piperidine in DMF for 12 min, washed 4× with DMF,4× with DCM and Fmoc-Phe(pNH-BOC)-OH was coupled using DIC/HOBt couplingin DMF. Deprotection of Fmoc and acetylation with aceticanhydride:pyridine (1:1) for 20 min gave the peptide resin,Ac-Phe(pNH-BOC)-Chg-Orn(C(NH)CH₃)-Leu-Pro-SCAL-TG. Reduction of the SCALlinker and cleavage of the peptide, followed by HPLC purification of thecrude product gave the expected compound. (M+H)⁺ found 740.2, calc.740.48.

EXAMPLE XXI Synthesis of Ac-Phe(pNH₂)-Chg-Dap(N^(β)—C₆H₁₁N)-Leu-Pro-NH₂

0.5 g SCAL-TG (0.32 mmol NH₂/g) was coupled with Fmoc-Pro-OH,Fmoc-Leu-OH, Fmoc-Dap(Boc)-OH and Fmoc-Chg-OH. The Boc group from theside chain was removed using 50% TFA for 20 min and the peptide resinwas neutralized by washing with 10% DIEA/DCM. The free amino group ofthe side chain was transformed to the dimethylamidinium group bytreatment of the peptide resin with 0.3 M PyBroP/NMI in DMF for 20 min.Fmoc group deprotection with 50% piperidine/DMF for 60 min resulted inexchange of the dimethylamidinium by the piperidinium group in the sidechain of Dap. The sequence was completed by coupling Fmoc-Phe(Boc)-OHand deprotection of the Fmoc group. The peptide was acetylated andcleaved as described in Example X. HPLC purified peptide was analyzed byMS. (M+H)⁺ found 752.4, calc. 752.4.

EXAMPLE XXII Synthesis of Ac-pAph-Chg-PalMe(3)-NH₂

Racemic H-Phe(pCN)-OH was synthesized by the acetamidomalonate method(Wagner et al., DDR Patent No. 155,954, issued Jul. 21, 1982; reexaminedNov. 9, 1988, which is incorporated herein by reference). The racemicAc-pAph-OH was synthesized by conversion of the cyano group byammonolysis of the corresponding methylthioimidate (offered by reactionof the cyano group with hydrogen sulfide) and subsequent methylation byMeI.

1 g “TENTAGEL” resin (substitution=0.21 mmol NH₂/g resin) and Knorrlinker (Bernatowicz et al., Tetr. Lett. 30:4645 (1989), which isincorporated herein by reference) were used for synthesis of thepeptide. The dipeptide, Fmoc-Chg-Pal-Knorr-TG, was assembled asdescribed in Example I. 3-pyridylalanine subsequently was methylated by1 ml MeI in DCM overnight. After Fmoc deprotection, Ac-pAph-OH wascoupled using the DIC/HOBt method and the peptide was worked up asdescribed in Example I. (M+H)⁺ found; 550.3, calc. 550.31.

EXAMPLE XXIII Synthesis of Ac-Tyr-Chg-pAph-Leu-Pro-NH₂

The pentapeptide, Ac-Tyr(But)-Chg-Phe(pCN)-Leu-Pro-Knorr-TG, wasassembled on 0.4 g “TENTAGEL” (substitution=0.2 mmol NH₂/g resin) asdescribed in Example I. The resin was treated overnight in a closedsyringe with 8 ml pyridine/triethylamine (75:25) saturated with H₂S. Theresin-bound thioamide was methylated using 0.5 ml MeI in 8 ml acetonefor 30 min at 50° C., then washed with acetone and methanol. Themethylthioimide was reacted with ammonium acetate in methanol for 3 hrat 55° C. to obtain the final compound, which was cleaved from the resinand purified as described above. (M+H)⁺ found 761.4, calc. 760.43.

EXAMPLE XXIV Synthesis of Ac-Phe(pCH₂NH₂)-Chg-Arg-Leu-Pro-NH₂

Ac-DL-Phe(pCN)-Chg-Arg-Leu-Pro-NH₂ (crude peptide) was synthesized on 1g Rink resin (0.6 mmol NH₂/g resin) as described in Example I. 125 mgcrude peptide was dissolved in 50 ml MeOH and 0.5 ml Raney Ni suspension(Aldrich) was added. The mixture of the peptide and catalyst washydrogenated at 35 psi for 4 hr at RT. The catalyst was filtered and thesolution was evaporated to dryness. The residue was lyophilized from0.1% aqueous TFA containing 30% ACN. The dried crude product waspurified by HPLC and analyzed by MS. (M+H)⁺ found 741.4, calc. 741.7.

EXAMPLE XXV Synthesis of Ac-Phe(pC(NOH)NH₂)-Chg-Arg-Leu-Pro-NH₂

21.1 mg crude peptide prepared as described in Example XXIV was mixedwith 60.3 mg NH₂OH.HCl (Aldrich) in 1.5 ml MeOH, 0.7 ml pyridine and 0.5ml TEA. The mixture was stirred for 72 hr at RT, then the solvent andvolatile materials were evaporated in a vacuum. The peptide was purifiedby HPLC and analyzed by MS. (M+H)⁺ found 770.4, calc. 770.3.

EXAMPLE XXVI Synthesis of A1—A2—B Compounds

A1—A2—B compounds, i.e., A1—A2—(A3)_(m)—B compounds in which m is 0,were prepared as outlined in shown in FIG. 3. Briefly, coupling ofracemic N-acetyl-4-cyanophenylalanine with L-cyclohexylglycine methylester, (H-Chg-OMe), yielded a mixture of two diastereomeric dipeptides,which were separated by chromatography. RacemicN-acetyl-4-cyanophenylalanine was partially resolved by forming the saltwith L-cyclohexylglycine methyl ester. The less soluble D,L-salt wascrystallized readily and subsequent coupling afforded theAc-f(pCN)-Chg-OMe in substantially pure form. The “mother liquors” wereenriched in the L,L-salt and coupling led to crude Ac-F(pCN)-Chg-OMe,which was further purified by chromatography over silica gel. Thesedipeptide esters were hydrolysed to the corresponding acids usinglithium hydroxide in methanol/water at RT. Both dipeptide acids wereconverted to the substituted amides by conventional coupling with theappropriate amines, RNH₂. The amines that were not commerciallyavailable were prepared using standard chemical methods.

Conversion of cyano groups to the corresponding amidines was performedusing standard chemical methods, either via the thioamide andmethylthioimidate or by hydrogenation of the corresponding amidoxime(Example XXV). The latter was obtained by reacting the nitrile withhydroxylamine. The examples described below illustrate the preparationof the title compounds by these selected methods. It is recognized thatcompounds of the invention can be prepared using various other methodsand that the procedures exemplified here were selected for convenience.

EXAMPLE XXVII Synthesis of Ac-pAoh-Chg-NHCH₂-(4-methylpyridinium)

Synthesis of Ac-pAph-Chg-NHCH₂-(4-methylpyridinium) was accomplished byconversion of Ac-F(pCN)-Chg-NHCH₂-(4-pyridyl) using the methodsdescribed in Example XXII. The final compound was purified by HPLC asdescribed in Example I. MS analysis: (M+H)⁺ found 493.3, calc. 493.29.

The starting material was prepared as follows:

a) Ac-(D,L)-F(pCN), 2.32 g (10 mmol) was dissolved in 75 ml ethanol bywarming. L-cyclohexylglycine methyl ester (1.75 g, 10 mmol) was addedand the mixture was stirred for 2 hr at RT. The precipitated crystalswere filtered off and dried to yield 1.55 g D,L-salt. The filtrate wasevaporated partially and diluted with ether. The separated crystals werecollected and dried to leave 2.1 g L,L-salt contaminated with D,L-salt.The crude L,L-salt was combined with 20 ml DMF, 0.71 g HOBt and 1.18 gDCC. The mixture was stirred 24 hr at RT. The urea was filtered off andthe filtrate was evaporated. The residue was dissolved in methylenechloride and the solution was washed with 1 N HCl and saturated aqueoussodium bicarbonate. The organic layer was dried and evaporated. Theresidue was chromatographed over 60 g silica gel using 20% (v/v) acetonein methylene chloride for elution. Crystallization of the combined cleanfractions from methylene chloride/ether/hexane gave 1.6 gAc-F(pCN)-Chg-OMe as colorless crystals with melting point (mp) of178-180° C.

b) A mixture of 1.93 g(5 mmol) Ac-F(pCN)-Chg-OMe (from Example XXVII.a.,above), 100 ml methanol, 10 ml water and 0.75 g lithium hydroxidehydrate was stirred under nitrogen for 24 hr at RT. Following additionof 2 ml acetic acid, the solvents were evaporated and the residue waspartitioned between methylene chloride containing 20% isopropanol and 1N HCl. The organic layer was dried and evaporated and the residue wascrystallized from methylene chloride/ether/hexane to leave 1.6 g ofAc-F(pCN)-Chg-OH as colorless crystals with mp 216-218° C.

c) A mixture of 150 mg (0.4 mmol) Ac-F(pCN)-Chg-OH (above), 65 mg (0.6mmol) 4-aminomethylpyridine, 124 mg (0.6 mmol) DCC, 60 mg (0.44 mmol)HOBt and 5 ml DMF was stirred for 20 hr at RT. The urea was removed byfiltration and the filtrate was evaporated. The residue was slurriedwith methanol and the insoluble product was collected by filtration toleave 140 mg colorless Ac-F(pCN)-Chg-NHCH₂(4-pyridyl). An analyticalsample was obtained by chromatography over silica gel usingacetone:methylene chloride:methanol (4:5:1). The crystalline solid hadmp>250° C.

EXAMPLE XXVIII

Ac-f(4-amidino)-Chg-NHCH₂(4-methylpyridinium)

This compound was prepared by reacting 150 mgAc-f(pCN)-Chg-NHCH₂(4-pyridyl) (see above) with hydrogen sulfide, thenwith methyl iodide and ammonium acetate. The product was isolated byHPLC as a homogenous material MS analysis: (M+H)⁺ found 493.3, calc.493.29.

The starting material was prepared as follows:

a) A mixture of 2.8 g Ac-f(pCN), (L)-cyclohexylglycine methyl ester, 940mg HOBt, 1.57 g DCC and 30 ml of DMF was stirred for 2 days at RT. Theurea was removed by filtration and the filtrate was evaporated. Theresidue was dissolved in methylene chloride and the solution was washedwith 1 N HCl and 10% aqueous sodium carbonate. The organic phase wasdried and evaporated. Crystallization of the residue from methylenechloride/ether/hexane gave 2.05 g colorless Ac-f(pCN)-Chg-OMe having amp 181-183° C.

b) Hydrolysis of 1.93 g Ac-f(pCN)-Chg-OMe (above) with 0.75 g lithiumhydroxide monohydrate in 100 ml methanol and 10 ml water was treated asdescribed for the L,L-isomer in Example XXVII, above, crystallized frommethylene chloride/ether, to produce 1.65 g Ac-f(pCN)-Chg-OH having a mp180-182° C.

c) A mixture of 225 mg Ac-f(pCN)-Chg-OH (above), 100 mg4-aminomethylpyridine, 90 mg HOBt, 180 mg DCC and 6 ml DMF was stirredover a weekend at RT. The urea was filtered off and the filtrate wasevaporated. The residue was stirred with methanol and the solids wereremoved by filtration to leave 190 mg crystallineAc-f(pCN)-Chg-NHCH₂(4-pyridyl) having a mp>250° C.

EXAMPLE XXIX Ac-pAph-Chg-NHCH₂CH₂(3-methylpyridinium)

A mixture of 125 mg of Ac-F(pCN)-Chg-NHCH₂CH₂(3-pyridyl), 2 ml DMSO, 10ml pyridine and 5 ml triethylamine was saturated with hydrogen sulfidewhile cooled in ice/water. After stirring in a sealed vial overnight atRT, the solvents were evaporated and the residue was collected withacetone/ether and dried to leave 125 mg of the thioamide. This materialwas combined with 2 ml DMSO, 5 ml acetone and 0.75 ml methyl iodide andthe mixture was stirred in a sealed vial overnight at RT. After dilutionwith toluene, the solvents were evaporated and the residue was stirredwith ether. The ether was decanted, replaced by fresh ether and stirringwas continued until the resinous material solidified, then the remainingether was filtered off and the residue dried.

The resulting residue was dissolved in 20 ml methanol and treated with0.3 ml acetic acid and 0.4 g ammonium acetate. The mixture was heated to55-60° C. for 2.5 hr, then solvents were evaporated. The residue wasdissolved in water/ACN/TFA and lyophilized. The crude product waspurified by HPLC. MS analysis: (M+H)⁺ found 507.3, calc. 507.31.

The starting material was obtained as follows. A mixture of 150 mg (0.4mmol) Ac-F(pCN)-Chg-OH, 120 mg (0.6 mmol) 2-(3-pyridyl)ethylaminedihydrochloride, 125 mg DCC, 60 mg HOBt, 0.5 ml diisopropylethylamineand 10 ml DMF was stirred for 24 hr at RT. After evaporation of thesolvent, the residue was stirred with methanol and the insoluble productwas collected by filtration and washed with methanol and ether to leave110 mg of colorless crystals. Thr filtrate was evaporated and theresidue was dissolved in methylene chloride/isopropanol. This solutionwas washed with 10% aqueous sodium carbonate, dried and evaporated. Theresidue was chromatographed over 14 g silica gel using methylenechloride:acetone:methanol (5:4:1) to yield 40 mgAc-F(pCN)-Chg-NHCH₂CH₂(3-pyridyl) having a mp 265-268° C.

b) 2-(3-pyridyl)ethylamine dihydrochloride was prepared as follows. Amixture of 1.3 g 3-pyridylacetonitrile, approximately 3 g Raney nickeland 30 ml methanol containing 10% ammonia by volume was hydrogenated at35 psi for 20 hr using a Parr hydrogenator. The catalyst was filteredoff over celite and the filtrate was evaporated. The residue wasdissolved in methylene chloride, dried with magnesium sulfate, filteredand evaporated. The product was converted to the dihydrochloride usinghydrogen chloride in dioxane. Crystallization from methanol/ether gave1.4 g colorless crystals having a mp 145-148° C.

EXAMPLE XXX Ac-pAph-Chg-NHCH₂CH₂(4-methylpyridinium)

This compound was prepared using methods as described above by reactingAc-F(pCN)-Chg-NHCH₂CH₂(4-pyridyl) with hydrogen sulfide followed bymethylation with methyl iodide and reaction with ammonium acetate. Thecrude product was purified by HPLC. MS analysis: (M+H)⁺ found 507.3,calc. 507.31.

The starting material was obtained by coupling of Ac-F(pCN)-Chg-OH with2-(4-pyridyl)ethylamine dihydrochloride as described in Example XXIX,above.

2-(4-pyridyl)ethylamine dihydrochloride prepared as described for2-(3-pyridyl)ethylamine dihydrochloride (above) by hydrogenation ofpyridyl-4-acetonitrile over Raney nickel in the presence of ammonia. Thedihydrochloride had a mp 220° C.

EXAMPLE XXXI Ac-pAph-Chg-NHCH₂(4-amidinophenyl)

This compound was prepared using similar methods as described above bytreating Ac-F(pCN)-Chg-NHCH₂(4-cyanophenyl) with hydrogen sulfide inDMSO, pyridine and triethylamine. The bis-thioamide obtained wasmethylated with methyl iodide in DMSO/acetone, then reacted withammonium acetate as described above. The crude product was purified byHPLC. MS analysis: (M+H)⁺ found 520.3, calc. 520.30.

The starting material was obtained as follows. A mixture of 75 mg (0.2mmol of Ac-F(pCN)-Chg-OH, 50 mg (0.3 mmol) (4-cyanophenyl)methylaminehydrochloride, 62 mg DCC, 30 mg HOBt, 0.2 ml DIEA and 2 ml DMF wasstirred for 24 hr at RT. After filtration, the solvent was evaporatedand the residue was dissolved in methylene chloride containing 20% ofisopropanol. The solution was washed with 1 N HCl and 10% aqueous sodiumcarbonate, then dried and evaporated. The residue was stirred with asmall amount methanol/water and the separated solids were collected anddried to leave 80 mg Ac-F(pCN)-Chg-NHCH₂(4-cyanophenyl).

(4-Cyanophenyl)methylamine hydrochloride was prepared as follows. Amixture of 2 g (10 mmol) α-bromo-p-tolunitrile, 2 g (10.8 mmol)potassium phthalimide and 30 ml DMF was heated to reflux for 1 min.After cooling, the mixture was acidified with acetic acid and dilutedwith water to crystallize the product. The crystals were filtered,washed with water and dried to leave 2.24 g colorlessN-(4-cyanophenyl)methylphthalimide having a mp 182-184° C.

1.5 g N-(4-cyanophenyl)methylphthalimide was suspended in 50 ml boilingmethanol and treated with 1 ml hydrazine hydrate. A clear solutionresulted after 5 min. The methanol was evaporated and the residue wastreated with 2 N HCl. The suspension was heated to boiling and thencooled on ice. The solids were filtered off and the filtrate wasevaporated. The residue was dissolved in water. The solution was heatedto boiling again, cooled and filtered. The filtrate was made alkalinewith sodium hydroxide and extracted with methylene chloride containingisopropanol. The organic phase was dried and evaporated and the residuewas converted to the hydrochloride salt, crystallized fromisopropanol/ether and yielded 0.43 g colorless crystals having a mp>260°C.

(3-Cyanophenyl)methylamine hydrochloride was prepared by reactingα-bromo-m-tolunitrile with potassium phthalimide to yieldN-(3-cyanophenyl)methylphthalimide having a mp 147-148° C. Reaction ofthis material with hydrazine hydrate and conversion to the hydrochlorideas above gave (3-cyanophenyl)methylamine having a mp 223-226° C.

EXAMPLE XXXII Ac-pAph-Chg-NHCH₂(3-amidinophenyl)

This compound was prepared using methods as described above.Ac-F(pCN)-Chg-NHCH₂(3-cyanophenyl) was treated with hydrogen sulfide inDMSO, pyridine and triethylamine. The bis-thioamide obtained wasmethylated with methyl iodide in DMSO/acetone, then reacted withammonium acetate as described above. The crude product was purified byHPLC. MS analysis: (M+S)⁺ found 520.3, calc. 520.30.

The starting material was obtained as follows. A mixture of 300 mg (0.8mmol) Ac-F(pCN)-Chg-OH, 200 mg (1.2 mmol) (3-cyanophenyl)methylaminehydrochloride, 250 mg DCC, 120 mg HOBt, 0.8 ml DIEA and 10 ml DMF wasstirred for 24 hr at RT. After filtration, the solvent was evaporatedand the residue was dissolved in a large volume of methylene chloridecontaining 20% isopropanol. The solution was washed with 1 N HCl and 10%aqueous sodium carbonate, dried and evaporated. The residue was stirredwith isopropanol/ether and the separated solids were collected and driedto leave 400 mg Ac-F(pCN)-Chg-NHCH₂(3-cyanophenyl).

EXAMPLE XXXIII Ac-pAph-Chg-NHCH(Me)(4-methylpyridinium)

A mixture of diastereomers of the title compound was prepared byreacting a mixture of two diastereomeric Ac-F(pCN)-Chg-NHCH(Me)(4-pyridyl) with hydrogen sulfide, then with MeI and ammonium acetate.The diastereomers were separated by HPLC. MS analysis: (M+H)⁺ found507.3, calc. 507.31.

The starting material was prepared as follows. A mixture of 150 mg (0.4mmol) Ac-F(pCN)-Chg-OH, 120 mg (0.6 mmol) racemic1-(4-pyridyl)ethylamine dihydrochloride, 125 mg DCC, 60 mg HOBt, 0.5 mlDIEA and 10 ml of DMF was stirred for 24 hr at RT. After filtration, thesolvent was evaporated and the residue was dissolved in a large volumeof methylene chloride containing 20% isopropanol. The solution waswashed with 10% aqueous sodium carbonate, dried and evaporated. Theresidue was stirred with isopropanol/ether and the separated solids werecollected and dried to leave 125 mg Ac-F(pCN)-Chg-NHCH(Me)(4-pyridyl) asa mixture of two diastereomers.

Racemic 1-(4-pyridyl)ethylamine dihydrochloride was prepared as follows.A mixture of 1 g 4-acetylpyridine-N-oxide, 2 g Raney nickel and 30 mlmethanol containing 20% ammonia (v/v) was hydrogenated for 24 hr at 30psi. The catalyst was removed by filtration over celite and the filtratewas evaporated. The residue was dissolved in methylene chloride,filtered and evaporated. The residue was dissolved in isopropanol andtreated with hydrogen chloride in ether. The precipitated crystals werecollected and dried to produce 0.9 g material having a mp 198-200° C.

EXAMPLE XXXIV Synthesis of DIPA(m)pAph-Chg-Arg-Leu-Pro-NH₂

a. Synthesis of N,N-Diisopropyl amide of (p-cyanobenzyl)malonicacid(DIPA(m)Phe(pCN))-OH

The synthesis of 2-(p-cyanobenzyl)malonic acid was achieved by amodified procedure (see Pinori et al, U.S. Pat. No. 5,061,911 (October,1991), which is incorporated herein by reference). To a solution of 3.8g 2,2-dimethyl-1,3-dioxane-4,6-dione (Meldrum's acid; Aldrich) and 1.12g NaCNBH₃ (Aldrich) in 25 ml DMF was added 2.3 g p-cyanobenzaldehyde(Aldrich) and the mixture was stirred 2 hr at RT. To the reactionmixture was added 400 ml water and the solution was cooled in an icebath and the pH was adjusted to 3.8-4 by dropwise addition of 20% HClaqueous solution. The white precipitate was collected in a centeredglass Buchner funnel and washed with cold water. The collectedprecipitate was dried in vacuo over CaCl₂ for 24 hr. The NMR of thecollected solid in CDCl₃ indicated the compound2,2-dimethyl-5-(p-cyano)benzyl-1,3-dioxane-4,6-dione (DCBD), which has amp 135-142° C. and R_(f) 0.45 (CHCl₃:MeOH:acetic acid; 95:4:1).

To 1.5 ml diisopropylamine in 45 ml DCM was added 3 mlN,O-Bis(trimethylsilyl)acetamide (BSA) and the solution was refluxed ina reaction flask equipped with a magnetic stirrer and a condenserguarded with a CaCl₂ tube for 7 hr. After cooling the solution to RT,0.8 g DCBD was added and the reaction mixture refluxed for 3 hr (untilcompletion of conversion to the product as indicated by TLC). Aftercooling the reaction mixture, 5-8 ml 20% HCl aqueous solution wascarefully added. After separation of the layers, the organic layer waswashed with water, dried (MgSO₄) and evaporated to dryness to give aclean product that was used in the next step without furtherpurification. The identification of the compounds was achieved by NMR inCDCL₃ and MS.

b. Synthesis of DIPA(m)pAph-Chg-Arg-Leu-Pro-NH₂

Peptide resin DIPA(m)Phe(pCN)-Chg-Arg(PMC)-Leu-Pro-Rink was synthesizedby the method described in Example I. The resulting peptide resin wastreated with hydroxylamine hydrochloride as described in Example XXV togive DIPA(m)Phe(pC(NOH)NH₂)-Chg-Arg(PMC)-Leu-Pro-Rink. After cleavage ofthe peptide from the resin and lyophilization, the crude product (120mg) was dissolved in 80 ml MeOH and 10 ml saturated solution of NH₃ inMeOH. To the reaction mixture was added 0.25 ml. Raney nickel suspension(Aldrich) and the mixture hydrogenated at 45 psi for 24 hr. The catalystwas filtered and the solvent evaporated to dryness and the residuelyophilized from 1:1 solution of 0.1% TFA aqueous solution and ACN. Thecrude peptide was purified by HPLC and the compound identified by MS.(M+H)⁺ found 824.2, calc. 824.5.

EXAMPLE XXXV Compounds with Multiple Substitutions that were Synthesizedand Found Potent Inhibitors of Factor Xa

No. Compound Calc. (Found) 1. Ac-(2-CF₃Bzl)—Y—I—R—L—P—NH₂ 860.5 (860.3)(SEQ ID NO:44) 2. Ac—(CH₃CH₂CH₂CH(CH₃)CH₂)—Y— 786.5 (786.5) I—R—L—P—NH₂(SEQ ID NO:43) 3. CH₃OCO—Y—I—R—L—P—NH₂ 742.4 (742.4) (SEQ ID NO:46) 4.Ac—Y—Chg—R—NH₂ 518.2 (518.2) 5. Nal(2)—Cha—R—D(O-Allyl)-NH₂ 679.4(679.4) 6. y-Tle—R—Nle—P—NH₂ 660.4 (660.4) 7. Phe(pF)—I—R—L—P—NH 662.3(662.3) 8. Ac—(D)Tic(OH)—I—R—L—P—NH₂ 714.4 (714.4) 9.Ac—Phe(pCN)—I—R—L—P—NH₂ 711.4 (711.4) (SEQ ID NO:47) 10.Ac—Phe(pCONH₂)—Chg—R—L—P—NH₂ 755.4 (755.4) 11. y-Chg—R—NH₂ 476.2 (476.2)12. Ac—W—Chg—R—L—P—NH₂ 751.3 (751.3) (SEQ ID NO:48) 13.Ac—Y—I—R—NH—CH(CH₃)—(CH₂)₂—CH₃ 562.3 (562.3) 14. Ac—Y—Pgl—R—L—P—NH₂722.2 (722.2) 15. Ac—Y—Chg—R—Ina—NH₂ 629.4 (629.4) 16. Ac—Tza—Chg—R—NH₂509.3 (509.3) 17. Ac—Y—Chg—R—Pip—NH₂ 629.4 (629.4) 18.Ac—Phe(pNH₂)—Chg—R—NH₂ 517.2 (517.2) 19. Ac—(Bzl)G—(Chx)Gly- 502.3(502.3) (3-guanidinopropyl)G—NH₂ 20. Ac—Y—Chg—R-ol.acetate 547.3 (547.3)21. Ac—Y—Chg—R—OCH₃ 533.3 (533.3) 22. Ac—Y—Chg—R—OH 519.3 (519.3) 23.Bz—Y—Chg—R—NH₂ 532.2 (532.2)

EXAMPLE XXXVI Combinations of Chemical Changes that Individually may nothave Improved Activity can Improve Activity

Inhibition of factor Xa activity was measured. However, any relevantmeasure of biological activity such as the effect of a YIR peptide ofthe invention on coagulation, in vivo potency, in vivo half-life, oralbioavailability, oral potency or half-life can be determined as ameasure of the activity of a peptide of the invention.

Many specific changes are depicted. As an example, two changes werecombined to demonstrate that a further improvement of activity wasobtained by combining changes, even where the original single changesdid not significantly improve activity. Single chemical changes producedAc-Y-I-R-L-P, which had a Ki=0.49 μM and (iBu)Y-I-R-L-P(SEQ ID NO: 50),which had a Ki=2.6 μM, compared to the parent compound, Y-I-R-L-P(SEQ IDNO: 51) (Ki=5.3 μM). Combining these two changes producedAc-(iBu)Y-I-R-L-P-NH₂, which had a Ki=0.04 μM. Thus, these resultsdemonstrate that a peptide of the invention having a combination of twochemical changes can have substantially increased factor Xa inhibitoryactivity as compared to the corresponding single change analogs, evenwhere one parent compound such as (iBu)Y-I-R-L-P-NH₂ did not have asignificantly improved activity compared to the parent Y-I-R-L-P-NH₂.

Table 3 exemplifies specific chemical modifications that resulted incompounds having K_(i) values between 100 μM and 1 pM for factor Xainhibition.

TABLE 3 Factor Xa Inhibitors having a Ki < 100 μM Structure MS* AA*(2,2-DiMe-Propyl)Y-I-R-L-P-NH₂ (SEQ ID NO:52) OK OK(2-CF₃-Bzl)Y—I—R—L—P—NH₂ (SEQ ID NO:53) OK OK (2-Et-nBu)Y—I—R—L—P—NH₂(SEQ ID NO:54) OK OK (2-Me-Bzl)Y—I—R—L—P—NH₂ (SEQ ID NO:55) OK OK(2-Me-nBu)Y—I—R—L—P—NH₂ (SEQ ID NO:56) OK OK (2-Me-nPentyl)Y—I—R—L—P—NH₂(SEQ ID NO:57) OK OK (3,3-DiMe-nBu)Y—I—R—L—P—NH₂ (SEQ ID NO:58) OK OK3-phenoxyproprionic-Y-Chg-R—NH₂ OK OK 5-Bzim-CO-Chg-R—L—P—NH₂ OK OK5-Bzim-CO—F(pNH₂)-Chg-R—L—P—NH₂ OK OK Y(3,5-Br)-I—R—L—P—NH₂ (SEQ IDNO:59) OK OK Y(3,5-I)—I—R—L—P—NH₂ (SEQ ID NO:60) OK OK(Chx-CH₂)Y—I—R—L—P—NH₂ (SEQ ID NO:61) OK OK (iBu)Y—I—R—OH OK OK(iBu)Y—I—R—L—A—NH₂ (SEQ ID NO:62) OK OK (iBu)Y—I—R—L—P—NH₂ (SEQ IDNO:63) OK OK (Me)y-I—R—L—A—NH₂ OK OK (Me)Y—I—R—L—A—NH₂ (SEQ ID NO:64) OKOK (Me)y-I—R—L—P—NH₂ OK OK (Me)Y—I—R—L—P—NH₂ (SEQ ID NO:65) OK OK5-Hic-Chg-R—NH₂ OK OK Ac-(1,2,3,6-4H-Bzl)Y(SO₃H)—I—R—L—P—NH₂ OK OK (SEQID NO:66) Ac-(1,2,3,6-4H-Bzl)Y—I—R—L—P—NH₂ OK OK (SEQ ID NO:67)Ac-(2,3-DiMe-nPentyl)Y(SO₃H)—I—R—L—P—NH₂ OK OK (SEQ ID NO:68)Ac-(2,3-DiMe-nPentyl)Y—I—R—L—P—NH₂ OK OK (SEQ ID NO:69)Ac-(2-CF₃-Bzl)Y(SO₃H)—I—R—L—P—NH₂ OK OK (SEQ ID NO:70)Ac-(2-CF₃-Bzl)Y—I—R—L—P—NH₂ (SEQ ID NO:71) OK OKAc-(2Et-nBu)Y—I—R—L—P—NH₂ (SEQ ID NO:72) OK OKAc-(2-Me-Bzl)Y(SO₃H)—I—R—L—P—NH₂ OK OK (SEQ ID NO:73)Ac-(2-Me-Bzl)Y—I—R—L—P—NH₂ (SEQ ID NO:74) OK OKAc-(2-Me-nBu)Y(SO₃H)—I—R—L—P—NH₂ OK OK (SEQ ID NO:75)Ac-(2-Me-nBu)Y—I—R—L—P—NH₂ (SEQ ID NO:76) OK OKAc-(2-Me-nPentyl)Y(SO₃H)—I—R—L—P—NH₂ OK OK (SEQ ID NO:77)Ac-(2-Me-nPentyl)Y—I—R—L—P—NH₂ (SEQ ID OK OK NO:78)Ac-(3,3-DiMe-nBu)Y(SO₃H)—I—R—L—P—NH₂ OK OK (SEQ ID NO:79)Ac-(3,3-DiMe-nBu)Y—I—R—L—P—NH₂ (SEQ ID OK OK NO:80)Ac-(3,3-DiMe-nPentyl)Y(SO₃H)—I—R—L—P—NH₂ OK OK (SEQ ID NO:81)Ac-(3,5,5-Me-3-nHexyl)Y—I—R—NH₂ OK OKAc-(3,5,5-Me-3-nHexyl)Y—I—R—L—P—NH₂ OK OK (SEQ ID NO:82)Ac-(4-pyridil-CH₂—)Y—I—R—NH₂ OK OK Ac-(4-MeO-Bzl)Y—I—R—NH₂ OK OKAc-(Bzl)Y—I—R—NH₂ OK OK Ac-(Chx-CH₂)Y—I—R—L—P—NH₂ (SEQ ID NO:83) OK OKAc-(Cyclopropyl-CH₂)Y(SO₃H)—I—R—L—P—NH₂ OK OK (SEQ ID NO:84)Ac-(Cyclopropyl-CH₂)Y—I—R—L—P—NH₂ OK OK (SEQ ID NO:85)Ac-(Et-CH═C(CH₃)—CH₂)Y(SO₃H)—I— OK OK R—L—P—NH₂ (SEQ ID NO:86)Ac-(Et-CH═C(CH₃)—CH₂)Y—I—R—L—P—NH₂ OK OK (SEQ ID NO:87)Ac-(iBu)F(pNH₂)-Chg-R—NH₂ OK OK Ac-(iBu)F(pNH₂)-Chg-R—L—P—NH₂ OK OKAc-(iBu-Nal(2)-Chg-R—L—P—NH₂ OK OK Ac-(iBu)Y-Chg-R—NH₂ OK OKAc-(iBu)Y-Chg-R—L—P—NH₂ OK OK Ac-(iBu)Y—I-Dab(N^(γ)—C₃H₇N)—L—P—NH₂ OK OKAc-(iBu)Y—I-Orn(N^(δ)—C₃H₇N)—L—P—NH₂ OK OK Ac-(iBu)Y—I—R—NH₂ OK OKAc-(iBu)Y—I—R—L—P—NH₂ OK OK Ac-(Me)Y-Chg-R—L—P—NH₂ OK OKAc-(Me)Y—I—R—L—A—NH₂ (SEQ ID NO:88) OK OK Ac-(Me)Y—I—R—L—P—NH₂ (SEQ IDNO:89) OK OK Ac-(nBu)Y—I—R—NH₂ OK OKAc-(trans-CH₃—CH═C(CH₃)—CH₂)Y—I—R—L—P— OK OK NH₂ (SEQ ID NO:90)Ac-Tyr(3,5-NO₂)—I—R—L—P—NH₂ (SEQ ID NO:91) OK OKAc-(Bzl)G-(Chx)Gly-(3-GuanidoPropyl)G—NH₂ OK OK Ac-βAla-Y—I—R—L—G—NH₂(SEQ ID NO:92) OK OK Ac-E—Y—I—R—L—K—NH₂** (SEQ ID NO:93) OK OKAc-E—Y—I—R—L—P—K—NH₂ (SEQ ID NO:94) OK OK Ac-F(pCONH₂)-Chg-R—L—P—NH₂ OKOK Ac-F(pCONH₂)—I—R—L—P—NH₂ (SEQ ID NO:95) OK OK Ac-F(pF)—I—R—L—P—NH₂(SEQ ID NO:96) OK OK Ac-f(pF)—I—R—L—P—NH₂ OK OK Ac-F(pCN)—I—R—L—P—NH₂(SEQ ID NO:97) OK OK Ac-F(pNH₂)-Chg-R—NH₂ OK OK Ac-F(pNH₂)-Chg-R—L—P—NH₂OK OK Ac-F(pNH₂)—I—R—L—P—NH₂ (SEQ ID NO:98) OK OKAc-F(pNH₂)-Chg-R-(Bzl)G—G—OH OK OK Ac-F(pNH₂)-Chg-R-(Chx)G—G—OH OK OKAc-F(pNH₂)-Chg-R—(CH₃CH₂CH₂ OK OK (CH₃))G—G—OH OK OK Ac-G—G—Y—I—R—G—NH₂(SEQ ID NO:99) OK OK Ac-G—Y-Nle-R—L—NH₂ (SEQ ID NO:100) OK OKAc-G-y-Nle-R—L—NH₂ OK OK Ac-G—Y—I—R—G—NH₂ (SEQ ID NO:101) OK OKAc-G—Y—I—R—L—NH₂ (SEQ ID NO:102) OK OK Ac-Nal(1)-I—R—L—P—NH₂ (SEQ IDNO:103) OK OK Ac-Nal(2)-Cha-R—D(O-Allyl)-NH₂ OK OKAc-Nal(2)-Cha-R—L—P—NH₂ OK OK Ac-Nal(2)-Chg-R—NH₂ OK OKAc-Nal(2)-Chg-R—L—P—NH₂ OK OK Ac-Nal(2)-I—R—L—P—NH₂ (SEQ ID NO:104) OKOK Ac-Pgl(OH)—I—R—L—NH₂ OK OK Ac-pAph-Chg-R—L—P—NH₂ OK OKAc-pAph-I—R—L—P—NH₂ (SEQ ID NO:105) OK OK Ac-Phe-(pGua)-I—R—L—P—NH₂ (SEQID NO:106) OK OK Ac-S—Y—I—R—L—P—NH₂ (SEQ ID NO:107) OK OKAc-W-Chg-R—L—P—NH₂ (SEQ ID NO:108) OK OK Ac-W—L—R—L—A—NH₂ (SEQ IDNO:109) OK OK Ac-Y(Me)-I—R—L—A—NH₂ (SEQ ID NO:110) OK OKAc-Y(Me)-I—R—L—P—NH₂ (SEQ ID NO:111) OK OK Ac-Y-(allo-I)-R—L—P—NH₂ (SEQID NO:112) OK OK Ac-Y-Cha-R—L—P—NH₂ (SEQ ID NO:113) OK OK Ac-Y-Chg-R—NH₂OK OK Ac-Y-Chg-R—NH—CH₂CH₂—N(CH₃)₄ OK OK Ac-Y-Chg-R—NH-Bzl-4-OMe OK OKAc-Y-Chg-R—NH—CH₂-Chx OK OK Ac-Y-Chg-R—NH—CH₂CH₂—N—(CH₃)₂ OK OKAc-Y-Chg-R—NH—CH₂CH₂—O—CH3 OK OK Ac-Y-Chg-R—NH—CH₂CH₂—COOH OK OKAc-Y-Chg-R—NH-Chx OK OK Ac-Y-Chg-R—NH—CH₂(2-(1-Et)pyrrolidinyl) OK OKAc-Y-Chg-R—NH—CH₂(2-(6-EtO)benzylthiazolyl) OK OK Ac-Y-Chg-R—L—P—NH₂(SEQ ID NO:114) OK OK Ac-Y-Chg-R(NO₂)-{Ψ(CH₂NH)}—L—NH₂ OK OKAc-Y-Nva-R—NH₂ OK OK Ac-Y-Pen(Me)-R—L—P—NH₂ (SEQ ID NO:115) OK OKAc-Y-Pgl-R—L—P—NH₂ (SEQ ID NO:116) OK OK Ac-Y-{Ψ(CH₂N(Ac))}-I—R—L—P—NH₂(SEQ OK OK ID NO:117) Ac-Y-{Ψ(CH₂NH)}-I—R—L—P—NH₂ (SEQ ID NO:118) OK OKAc-Y—I—R-{Ψ(COCH₂)}-G—P—NH₂ (SEQ ID NO:119) OK OKAc-Y—I-Dab(N^(γ)C₃H₇N)-L—A—NH₂ (SEQ ID NO:120) OK OK Ac-Y—I-hR-L—A—NH₂(SEQ ID NO:121) OK OK Ac-Y—I-nR-L—A—NH₂ (SEQ ID NO:122) OK OKAc-Y—I-PalMe(3)-NH₂ OK OK Ac-Y—I-PalMe(3)-L-P-NH₂ (SEQ ID NO:123) OK OKAc-Y—I-{Ψ(CH₂NH)}-R—L—P—NH₂ (SEQ ID NO:124) OK OK Ac-y-I—R—NH₂ OK OKAc-Y—I—R—NH₂ OK OK Ac-Y—I—R—N(Me)O—CH₃ OK OK Ac-Y—I—R—NH—CH₂-4-PyridylOK OK Ac-Y—I—R—NH—CH₂CH₂—N(CH₃)₂ OK OK Ac-Y—I—R—NH-4-morpholinyl OK OKAc-Y—I—R—NH—OCH3 OK OK Ac-Y—I—R-Nle-Hyp OK OK Ac-Y—I—R-Nle-Δ²P OK OKAc-Y—I—R-Piperidyl OK OK Ac-Y—I—R—I (SEQ ID NO:125) OK OK Ac-Y—I—R—I—P(SEQ ID NO:126) OK OK Ac-Y—I—R—L (SEQ ID NO:127) OK OK Ac-y-I—R—L—A OKOK Ac-Y—I—R—L—A OK OK Ac-Y—I—R—L—A—A—F—T—NH₂ (SEQ ID NO:128) OK OKAc-y-I—R—L—P—NH₂ OK OK Ac-Y—I—R—L—P—NH₂ OK OKAc-Y—I—R—L—P—Dab(Ac-Y—I—R—L— OK OK P(G-A)₃)-OHAc-Y—I—R—L—P—Dab(Ac-Y—I—R—L— OK OK P(G-A)₆)-OHAc-Y—I—R—L—P—Dab(Ac-Y—I—R—L—P)—OH OK OK Ac-Y—I—R—L—P—NH₂ (SEQ ID NO:129)OK OK Ac-Y—K—R—L—E—NH₂ (SEQ ID NO:130) OK OK Ac-Y—N—R—L—NH₂ (SEQ IDNO:131) OK OK Ac-Y—N—R—L—P—NH₂ (SEQ ID NO:132) OK OKAc-Y—T(Me)-R—L—P—NH₂ (SEQ ID NO:133) OK OK βAla-Y—I—R—G (SEQ ID NO:231)OK OK βAla-Y—I—R—G—NH₂ (SEQ ID NO:232) OK OK Caff-I—R—NH₂ OK OKCbz-I—R—L—NH₂ OK OK Cbz-I—R—NH₂ OK OK CClF₂—CO—Y—I—R—L—P—NH₂ (SEQ IDNO:134) OK OK CF₂H—CO—Y—I—R—L—P—NH₂ (SEQ ID NO:135) OK OKCF₃—CF₂CO—Y—I—R—L—P—NH₂ (SEQ ID OK OK NO:136) CH₃—CHCl—CO—Y—I—R—L—P—NH₂(SEQ ID OK OK NO:137) CH₃O—CO—Y—I—R—L—P—NH₂ (SEQ ID NO:138) OK OKCH₃—SO₂—Y—I—R—L—P—NH₂ (SEQ ID NO:139) OK OK CH₃CH₂—O—CO—Y—I—R—L—P—NH₂ OKOK (SEQ ID NO:140) Cl₂CHCO—Y—I—R—L—P—NH₂ (SEQ ID NO:141) OK OKClCH₂CO—Y—I—R—NH₂ OK OK C—Y—I—R—L—C—NH₂ (SEQ ID NO:142) OK OKD-Tic-I—R—L—A—A—F—T—NH₂ (SEQ ID NO:143) OK OK Et(Et)Y—I—R—L—P—NH₂ (SEQID NO:144) OK OK E—Y—I—R—K—NH₂ (SEQ ID NO:145) OK OK E—Y—I—R—L—K—NH₂(SEQ ID NO:146) OK OK E—Y—I—R—L—P—K—NH₂ (SEQ ID NO:147) OK OKF(pCl)—I—R—I-Sar-NH₂ OK OK F(pF)—I—R—L—P—NH₂ (SEQ ID NO:148) OK OKf(pF)—I—R—L—P—NH₂ OK OK F(pNH₂)—I—R—L—A—NH₂ (SEQ ID NO:149) OK OKF(pNO₂)—I—R—L—A—NH₂ (SEQ ID NO:150) OK OK f-I—R—F—P—NH₂ OK OKf-I—R—I—P—NH₂ OK OK F—I—R—L—N—H₂ (SEQ ID NO:151) F—I—R—L—P—H—Y—G—NH₂(SEQ ID NO:152) OK OK F—I—R—L—Y—V—W—N—NH₂ (SEQ ID NO:153) OK OKFor-y-I—R—L—P—NH₂ OK OK For-Y—I—R—L—P—NH₂ (SEQ ID NO:154) OK OKG—G—Y—I—R—G—NH₂ (SEQ ID NO:155) OK OK G—Y—I—R—D—NH₂ (SEQ ID NO:156) OKOK G—Y—I—R—F—NH₂ (SEQ ID NO:157) OK OK G—Y—I—R—G—NH₂ (SEQ ID NO:158) OKOK G—Y—I—R—G (SEQ ID NO:159) OK OK G—Y—I—R—H—NH₂ (SEQ ID NO:160) OK OKG—Y—I—R—I—NH₂ (SEQ ID NO:161) OK OK G—Y—I—R—K—NH₂ (SEQ ID NO:162) OK OKG—Y—I—R—L—NH₂ (SEQ ID NO:163) OK OK G—Y—I—R—L—P—NH₂ (SEQ ID NO:164) OKOK G—Y—I—R—L—P—A—M—NH₂ (SEQ ID NO:165) OK OK G—Y—I—R—L—P—P—V—NH₂ (SEQ IDNO:166) OK OK G—Y—I—R—L—P—Q—T—NH₂ (SEQ ID NO:167) OK OKG—Y—I—R—L—P—S—Q—NH₂ (SEQ ID NO:168) OK OK G—Y—I—R—S—NH₂ (SEQ ID NO:169)OK OK G—Y—I—R—T—NH₂ (SEQ ID NO:170) OK OK G—Y—I—R—V—NH₂ (SEQ ID NO:171)OK OK G—Y—I—R—W—NH₂ (SEQ ID NO:172) OK OK G—Y—I—R—Y—NH₂ (SEQ ID NO:173)OK OK (pOH)C₆H₄—CH₂CH₂(OH)—CO—I—R—L-Sar-NH₂ OK OK(pOH)C₆H₄—CH₂CH₂CO—I—R—L—A—NH₂ OK OK (SEQ ID NO:174)(pOH)C₆H₄—CH₂CH₂CO—I—R—L—P—NH₂ OK OK (SEQ ID NO:175)(pOH)C₆H₄—CH₂CHOH—CO—I—R—L—P—NH₂ OK OK (SEQ ID NO:176)(pOH)C₆H₄—OCH(CH₃)CO—I—R—L—P—NH₂ OK OK (SEQ ID NO:177)(pOH)C₆H₄—OCH₂CO—I—R—L—P—NH₂ OK OK (SEQ ID NO:178) I—H—L—W—Y—I—R—L—P—NH₂(SEQ ID NO:179) OK OK I—H—L—W-y-I—R—L—P—NH₂ OK OK I—Q—L—G—Y—I—R—L—P—NH₂(SEQ ID NO:180) OK OK 4-MeO-C₆H₄—CO—I—R—L—A—NH₂ (SEQ ID OK OK NO:181)N-morpholinyl-CO—F—I—R—L—P—NH₂ OK OK (SEQ ID NO:182)Nal(2)-Cha-R—D(O-Allyl)-NH₂ OK OK Nal(2)-Cha-R—D(O-Allyl)-Sar-NH₂ OK OKNal(2)-Chg-R—L—P—NH₂ OK OK Nal(2)-I—R—C(Me)—P—NH₂ OK OKN—G—Y—I—R—L—I—H—NH₂ (SEQ ID NO:183) OK OK pal-C(SBut)-R—L—P—NH₂ OK OKpal-I—R—C(SBut)-Hyp-NH₂ OK OK pal-I—R—C(SBut)-P—NH₂ OK OKPgl(OH)—I—R—L—NH₂ OK OK Ph-C(NOCH₂Ph)-CO—I—R—NH₂ OK OKPh-CH═CH—CO—I—R—L—A—NH₂ (SEQ ID NO:184) OK OKPh-CH₂CH₂CH₂—CO—I—R—L—A—NH₂ OK OK (SEQ ID NO:185)Ph-CH₂CH₂CO—I—R—L—A—NH₂ (SEQ ID NO:186) OK OK Pth-Y—I—R—L—P—NH₂ (SEQ IDNO:187) OK OK S—Y—I—R—L—P—NH₂ (SEQ ID NO:188) OK OKTfa-(iBu)F(pNH2)-Chg-R—NH₂ OK OK Tfa-(iBu)F(pNH₂)-Chg-R—L—P—NH₂ OK OKTfa-(iBu)Nal(2)-Chg-R—L—P—NH₂ OK OK Tfa-(iBu)Y-Chg-R—NH₂ OK OKTfa-(iBu)Y-Chg-R—L—P—NH₂ OK OK Tfa-(iBu)Y—I-Dab(N^(γ)—C₃H₇N)—L—P—NH₂ OKOK Tfa-(iBu)Y—I-Orn(N^(δ)—C₃H₇N)—L—P—NH₂ OK OK Tfa-(iBu)Y—I-PalMe(3)-NH₂OK OK Tfa-(iBu)Y—I—R—NH₂ OK OK Tfa-(iBu)Y—I—R—OH OK OKTfa-(iBu)Y—I—R—G—NH₂ OK OK Tfa-(iBu)Y—I—R—L—P—NH₂ (SEQ ID NO:189) OK OKTfa-(Me)Y—I—R—L—A—NH₂ (SEQ ID NO:190) OK OK Tfa-Y(Me)I—R—L—P—NH₂ (SEQ IDNO:191) OK OK Tfa-Y-Chg-R—NH₂ OK OK Tfa-Y-Chg-R—L—P—NH₂ (SEQ ID NO:192)OK OK Tfa-y-I—R—L—P—NH₂ OK OK Tfa-Y—I—R—L—P—NH₂ (SEQ ID NO:193) OK OKT—F—G—Y—I—R—K—A—NH₂ (SEQ ID NO:194) OK OK Tos-Y—I—R—NH₂ OK OKTos-G—I—R—V-Sar-NH₂ (SEQ ID NO:195) OK OK Tyr(Me)-I—R—L—A—NH₂ (SEQ IDNO:196) OK OK W—F—R—E—M—G—G—G—G—G—NH₂ OK OK (SEQ ID NO:197)W—I—R—E—K—NH₂ (SEQ ID NO:198) OK OK W—I—R—N—P—NH₂ (SEQ ID NO:199) OK OKW—I—R—T—P—NH₂ (SEQ ID NO:200) OK OK w-L—R—L—A—NH₂ OK OK W—L—R—L—A—NH₂(SEQ ID NO:201) OK OK W—L—R—L—A—G—G—G—G—G—NH₂ OK OK (SEQ ID NO:202)W—L—R—V—A—NH₂ (SEQ ID NO:203) OK OK w-L—R—V—A—NH₂ OK OKW—L—R—V—A—G—G—G—G—G—NH₂ OK OK (SEQ ID NO:204) y(Me)-I—R—L—P—NH₂ OK OKy-Chg-R—NH₂ OK OK y-Chg-R—L—NH₂ OK OK y-Chg-R—L—P—NH₂ OK OKy-Tle-R-Nle-P—NH₂ OK OK y-Tle-R-Nle-Δ²P—NH₂ OK OK y-I-(nR)-L—A—NH₂ (SEQID NO:205) OK OK y-I—R(COCH2)—G—P—NH₂ OK OK y-I—R—NH₂ OK OK Y—I—R—NH₂ OKOK Y—I—R—E—F—S—D—Y—NH₂ (SEQ ID NO:206) OK OK Y—I—R—G—A—NH₂ (SEQ IDNO:207) OK OK Y—I—R—I—NH₂ (SEQ ID NO:208) OK OK Y—I—R—I—Y—NH₂ (SEQ IDNO:209) OK OK Y—I—R—I—Y—E—R—E—NH₂ (SEQ ID NO:210) OK OK Y—I—R—L—NH₂ (SEQID NO:211) OK OK y-I—R—L—A—NH₂ OK OK Y—I—R—L—a—NH₂ OK OK Y—I—R—L—A—NH₂(SEQ ID NO:212) OK OK Y—I—R—L—A—A—NH₂ (SEQ ID NO:213) OK OKY—I—R—L—A—A—F—NH₂ (SEQ ID NO:214) OK OK Y—I—R—L—A—A—F—T—NH₂ (SEQ IDNO:215) OK OK Y—I—R—L—M—E—M—T—NH₂ (SEQ ID NO:216) OK OK y-I—R—L—P—NH₂ OKOK Y—I—R—L—P—NH₂ (SEQ ID NO:217) OK OK Y—I—R—L—P—G—L—L—NH₂ (SEQ IDNO:218) OK OK Y—I—R—L—T—K—M—W—NH₂ (SEQ ID NO:219) OK OKY—I—R—V—A—Q—L—Y—NH₂ (SEQ ID NO:220) OK OK Y—I—R—V—M—N—H—R—NH₂ (SEQ IDNO:221) OK OK Y—I—R—Y—R—N—P—I—NH₂ (SEQ ID NO:222) OK OKY—R—Y—P—R—D—R—N—NH₂ (SEQ ID NO:223) OK OK Y—L—R—F—P—NH₂ (SEQ ID NO:224)OK OK *MS, mass spectrometry; AA, amino acid analysis. **  underliningindicates cyclized portion of peptide.

EXAMPLE XXXVII In vitro Inhibition of Selected Purified CoagulationEnzymes and Other Serine Proteases

The ability of a compound of the invention to inhibit factor Xa,thrombin, plasmin, elastase and trypsin was assessed by determining theconcentration of YIR peptide that inhibits enzyme activity by 50%(IC₅₀). Purified enzymes were used in chromogenic assays. To determinethe inhibition constant, the IC₅₀ value was corrected for competitionwith substrate using the formula:

Ki=IC₅₀×(1/{1+((substrate concentration)/substrate Km)})

(Chen and Prusoff, Biochem. Pharmacol. 22:3099-3018 (1973), which isincorporated herein by reference).

a. Factor Xa Assay

TBS-P buffer (50 mM Tris-Cl, pH 7.8, 200 mM NaCl, 0.05% (w/v) PEG-8000,0.02% (w/v) NaN₃) was used for this assay. The IC₅₀ was determined bycombining in appropriate wells of a Costar half-area microtiter plate 25μl human factor Xa (Enzyme Research Laboratories, Inc.; South Bend Ind.)in TBS-P; 40 μl 10% (v/v) DMSO in TBS-P (uninhibited control) or variousconcentrations of a peptide to be tested diluted in 10% (v/v) DMSO inTBS-P; and substrate S-2765(Nα-benzyloxycarbonyl-D-Arg-Gly-L-Arg-p-nitroanilide; Kabi Pharmacia,Inc.; Franklin Ohio) in TBS-P.

The assays were performed by pre-incubating the peptide inhibitor plusenzyme for 10 min, then the assay was initiated by adding substrate toobtain a final volume of 100 μl. The initial velocity of chromogenicsubstrate hydrolysis was measured by the change in absorbance at 405 nMusing a Bio-tek Instruments kinetic plate reader (Ceres UV900HDi) at 25°C. during the linear portion of the time course (usually 1-5 min afteraddition of substrate). The concentration of inhibitor that caused a 50%decrease in the rate of substrate hydrolysis was predicted by linearregression after plotting the relative rates of hydrolysis (compared tothe uninhibited control) versus the log of the peptide concentration.The enzyme concentration was 0.5 nM and substrate concentration was 140μM.

b. Thrombin Assay

TBS-P buffer was used for this assay. The IC₅₀ was determined asdescribed in Example XXXVII.a., except that the substrate was S-2366(L-PyroGlu-L-Pro-L-Arg-p-nitroanilide; Kabi) and the enzyme was humanthrombin (Enzyme Research Laboratories, Inc.; South Bend Ind.). Theenzyme concentration was 1 nM and substrate concentration was 175 μM.

c. Plasmin Assay

TBS-P buffer was used for this assay. The IC₅₀ was determined asdescribed in Example XXXVII.a., except that the substrate was S-2251((D)-Val-L-Leu-L-Lys-p-nitroanilide; Kabi) and the enzyme was humanplasmin (Kabi). The enzyme concentration was 5 nM and the substrateconcentration was 300 uM.

d. Trypsin Assay

TBS-P buffer containing 10 mM CaCl2 was used for this assay. The IC₅₀determination was determined as described in Example XXXVII.a., exceptthat the substrate was BAPNA (Benzoyl-L-Arg-p-nitroanilide; SigmaChemical Co.; St. Louis Mo.) and the enzyme was bovine pancreatictrypsin (Type XIII, TPCK treated; Sigma). The enzyme concentration was50 nM and the substrate concentration was 300 μM.

e. Elastase Assay

Tris-Cl, pH 7.4, 300 mM NaCl, 2% (v/v) N-methyl-pyrrolidone, 0.01% (w/v)NaN3 buffer was used for this assay. The IC₅₀ was determined asdescribed in Example XXXVII.a., except that the substrate wassuccinyl-Ala-Ala-Ala-p-nitroanilide (Calbiochem-Nova Biochem Corp.; SanDiego Calif.) and the enzyme was human neutrophil elastase (AthensResearch and Technology, Inc.; Athens Ga.). The enzyme concentration was75 nM and the substrate concentration was 600 μM.

The Ki values for selected test compounds compared to the controlcompound “TENSTOP” (N-alpha-tosyl-Gly-p-amidinophenylalanine methylester; American Diagnostica, Inc.; Greenwich Conn.), which is areversible factor Xa inhibitor (Sturzebecher et al., Thromb. Res.54:245-252 (1989); Hauptmann et al., Thromb. Haem. 63:220-223 (1990),each of which is incorporated herein by reference) are shown in Table 2,above. The results demonstrate that the YIR peptides of the inventioncan inhibit factor Xa activity but do not substantially inhibit theactivity of various other serine proteases, including thrombin andplasmin, which are involved in the process of blood coagulation andfibrinolysis.

EXAMPLE XXXVIII Assays for Determining Inhibition of Coagulation

The compounds of the invention were assessed for their ability toinhibit factor Xa activity. Effectiveness of various compounds wasassessed by the in vitro prothrombin time (PT) assay using pooled humandonor plasma. An ex vivo assay also was used in which plasma wascollected at various times after intravenous (iv) administration of acompound to rats and to rabbits or intraduodenal administration to ratsand analyzed using the PT assay to determine plasma half-life. The PTassay was initiated with a thromboplastin dilution selected to obtain anextended and highly reproducible coagulation endpoint, referred to asthe “dilute PT assay” as described below. Effectiveness of variouscompounds also was determined using an in vivo rat arteriovenous shuntmodel of thrombosis.

a. In Vitro Dilute Prothrombin Time Assay

100 μl prewarmed (37° C.) pooled human platelet poor plasma (PPP) wasadded to a fibrometer cup (Baxter Diagnostics., Inc.; McGaw Park Ill. 50μl of various concentrations of a test compound in TBS-BSA with calcium(50 mM Tris-Cl, 100 mM NaCl, 0.1% (w/v) bovine serum albumin, 20 mMCaCl₂) was added. In control experiments, TBS-BSA with calcium butwithout test compound was added for measurement of uninhibitedcoagulation time. 150 μl diluted prewarmed rabbit thromboplastin(Baxter) with calcium was added to the fibrometer cup and the fibrometertimer is started. A rabbit thromboplastin dilution curve was obtainedprior to testing the compound and is used to choose a thromboplastindilution that allows approximately 30 sec PT time for uninhibitedcontrols. The experimental concentration giving 50% inhibition ofcoagulation (EC₅₀) with test compound (see Table 4, below) wascalculated from the dilution curve times.

Alternatively, the dilute prothrombin time assay was conducted using the“research” mode on an Instrumentation Laboratories (IL) ACL3000-plusautomated coagulation instrument (IL; Milan, Italy). Thromboplastin wasdiluted until a clotting time of 30-35 seconds was achieved. Thisclotting time was taken as 100% activity. A standard curve forcalibration was established by serial 2-fold dilution of the dilutedthromboplastin reagent (rabbit brain IL-brand thromboplastin). Duringthe assay, a 50 μl sample (plasma separated by centrifugation) was mixedwith 100 μl thromboplastin reagent and nephelometric readings were takenover 169 sec. Coagulation time was determined from the maximal rate ofchange of light scatter calculated by the instrument. Inhibition isexpressed as percent activity as determined by comparison with thecalibration curve.

b. Ex Vivo Dilute Prothrombin Time Assay

Test compound was administered iv either through the tail vein (rat) orear vein (rabbit) following an approved protocol. One ml blood sampleswere removed at timed intervals after administration of a test compoundfrom a cannulated carotid artery (rat) or auricular artery (rabbit).After centrifugation to obtain PPP, the plasma was immediately stored onice or frozen.

For dilute prothrombin time determination, the plasma was prewarmed andassayed as described above. Percent inhibition was calculated from athromboplastin dilution curve, which was run with each series ofsamples, and used to determine the time at which approximately 50% ofthe initial anticoagulant activity remains in the plasma (T1/2). Theresults of this experiment demonstrate that the YIR peptides of theinvention can inhibit blood coagulation in in vitro and afteradministration in vivo (see Table 4).

TABLE 4 Activities and half-lives of selected inhibitors EC₅₀ PooledT_(1/2) Human Rat Rabbit Structure in vitro ex vivo ex vivoAc—Y—Chg—R—NH₂  2.5 nM 5 min 5 min Ac—Y—Chg—R—L—P—NH₂ 225 nM 5 min 5 minAc—Nal(2)—Chg—R—L—P—NH₂ 140 nM 6 min 5 min Tfa—(iBu)Y—Chg—R—L—P—NH₂ 300nM 15 min  10 min 

Various compounds also were examined for anticoagulant activity usingthe ex vivo dilute prothrombin time assay following iv bolusadministration of various doses in rats. The compounds listed in Table 5demonstrated at least 30% inhibition 10 min after administration of ≦2mg/kg of the indicated compound. These results demonstrate that variousrepresentative YIR peptides of the invention have substantialanticoagulant activity. The structures of all of the compounds listed inTable 5 were confirmed by MS and AA.

TABLE 5 1. Ac—pAph—Chg—PalMe(3)—NH—CH₂—Chx 2.Ac—pAph—Chg—PalMe(3)—NH—2CMT 3. Ac—pAph—Chg—PalMe(3)—NH—Chx 4.Ac—F(pNH₂)—Chg—Dab(N^(γ)—C₃NH₇)L—P—NH₂ 5. Bz—F(pNH₂)—Chg—R—L—P—NH₂ 6.Tos—F(pNH₂)—Chg—R—L—P—NH₂ 7. Ac—Y(3—I)—Chg—R—L—P—NH₂ 8.Ac—pAph—Chg—AMP(4) 9. y—Chg—R—L—NH₂ 10. Ac—F(pNH₂)—Chg—R—ol 11.Cyclopentyl-CO—pAph—Chg—PalMe(3)—NH₂ 12. 3—Iqc—pAph—Chg—PalMe(3)—NH₂ 13.Bzf—pAph—Chg—PalMe(3)—NH₂ 14. 3—Iqc—F(pNH₂)—Chg—R—L—P—NH₂ 15.Ac—F(pNH₂)—Chg—R-Thiazolyl 16. 2-Furoyl—pAph—Chg—PalMe(3)—NH₂ 17.5-Me-thienyl-CO—pAph—Chg—PalMe(3)—NH₂ 18. Ac—Nal(2)—Chg—R-Thiazolyl 19.2-Bzf—f(pNH₂)—Chg—R—L—P—NH₂ 20. Ac—pAph—Chg—Dab(N^(γ)—C₃NH₇)—L—P—NH₂ 21.Ac—Orn—Nal—(2)—Chg—PalMe(3)—Sar—E—NH₂* 22.Ac—Phe(3-I,4-NH₂)—Chg—R—L—P—NH₂ 23. Ac—(iBu)pAph—Chg—R—L—P—NH₂ 24.Ac—pAph—Chg—R—Gla—P—NH₂ 25. Ac—pAph—Chg—R—Pen(CH₂COOH)—P—NH₂ 26.Ac—pAph—Chg—R—L—P—NH₂ 27. Ac—F(pNH₂)—Chg—R—(Me)L—P—NH₂ 28.Ac—F(pNH₂)—Chg—R—OEt 29. Ac—F(pNH₂)—Chg—Orn(N^(δ)—C₃H₇N)—L—P—NH₂ 30.Ac—F(pNH₂)—Chg—R—L—P—NH₂ 31. Ac—Nal(2)—Chg—R—L—P—NH₂ 32.Ac—pAph—Chg—Dab(N^(γ)—C₃H₇N) 33. Ac—pAph—Chg—PalMe(3)—NH₂ 34.Ac—pAph—Chg—PalMe(3)—L—P—NH₂ 35. Ac—pAph—Chg—R—NH₂ 36. Ac—pAph—Chg—R—OH37. Ac—Y—Chg—R—NH—Nip—NH₂ 38. Ac—K—Nal(2)—Chg—R—Hyp—E—NH₂ 39.DIPA—pAph—Chg—R—L—P—NH₂ 40. DIPA—mF(pNH₂)—Chg—R—L—P—NH₂ 41.Isn—F(pNH₂)—Chg—R—L—P—NH₂ 42. Pza—F(pNH₂)—Chg—R—L—P—NH₂ 43.Tfa—(iBu)F(pNH₂)—Chg—R—L—P—NH₂ 44. Tfa—(iBu)Y—Chg—R—L—P—NH₂ 45.Tfa—(iBu)Y—I—Orn(N^(δ)—C₃H₇N)—L—P—NH₂ *underlining indicates cyclicportion of compound.

In some experiments, the test compounds were administered to rats usingan intraduodenal dosing protocol. Male Sprague-Dawley rats weighingapproximately 300 g were anesthetized with a combination ofketamine/xylazine, subcutaneously, following an approved protocol. Theright carotid artery was cannulated for blood sampling. A laparotomy wasperformed and duodenum was cannulated with a ball-tip needle and tiedinto place to ensure that the suture was distal to the point ofinsertion. An additional tie was placed proximal to the insertion pointto prevent leakage of gastric contents. The effectiveness of the suturein preventing a compound from reaching the site of insertion was testedby pressure testing at the conclusion of each experiment. The point ofinsertion was approximately 4 cm from the duodenal-gastric junction.Compounds were administered in 1 ml normal saline. A 0.7 ml blood samplewas drawn prior to administration of the test compound and at 15, 30,60, 90 and 120 min after administration. Plasma was separated bycentrifugation and assayed for inhibition of coagulation using thedilute prothrombin time assay.

The following compounds showed at least 30% inhibition in the diluteprothrombin time assay following intraduodenal administration of ≦50mg/kg compound: Ac-pAph-Chg-PalMe(3)-NH—CH₂-Chx;Ac-pAph-Chg-PalMe(3)-NH-Chx; Bzf-pAph-Chg-PalMe(3)-NH₂;Ac-F(pNH₂)-Chg-R-L-P-NH₂; Ac-pAph-Chg-PalMe(3)-L-P-NH₂;Ac-pAph-Chg-PalMe(3)-NH₂; Ac-Aph-Chg-AMP(4);Cyclopentyl-CO-pAph-Chg-PalMe(3)-NH₂; 3-Iqc-pAph-Chg-PalMe(3)-NH₂;2-Furoyl-pAph-Chg-PalMe(3)-NH₂; 5-Me-thienyl-CO-pAph-Chg-PalMe(3)-NH₂,Ac-Y(3-I)-Chq-R-L-P-NH₂, Ac-F(pNH₂)-Chg-R-ol andAc-pAph-Chg-PalMe(3)-ol.

c. Rat Arteriovenous Shunt Model of Thrombosis

The anti-thrombotic efficacy of various compounds of the invention wasassessed using rat extracorporeal arteriovenous (AV) shunt. The AV shuntcircuit consisted of a 20 cm length of polyethylene (PE) 60 tubinginserted into the right carotid artery, a 6 cm length of PE 160 tubingcontaining a 6.5 cm length of mercerized cotton,thread (5 cm exposed toblood flow), and a second length of PE 60 tubing (20 cm) completing thecircuit into the left jugular vein. The entire circuit was filled withnormal saline prior to insertion.

Test compounds were administered by continuous infusion into the tailvein using a syringe pump and butterfly catheter (infusion volume 1.02ml/hr). A compound was administered for 30 min, then the shunt wasopened and blood was allowed to flow for a period of 15 min (total of 45min infusion). At the end of the 15 min period, the shunt was clampedand the thread was carefully removed and weighed on an analyticalbalance. Percent inhibition of thrombus formation was calculated usingthe thrombus weight obtained from control rats, which were infused withsaline.

The following compounds inhibited thrombus growth by at least about 30%following an infusion of ≦33 μg/kg/min: Ac-pAph-Chg-PalMe(3)-NH—CH₂-Chx;Ac-pAph-Chg-PalMe(3)-NH-Chx; Bzf-pAph-Chg-PalMe(3)-NH₂;Ac-pAph-Chg-PalMe(3)-L-P-NH₂; Ac-pAph-Chg-PalMe(3)-NH₂;Ac-pAph-Chg-AMP(4); Cyclopentyl-CO-pAph-Chg-PalMe(3)-NH₂;3-Iqc-pAph-Chg-PalMe(3)-NH₂; 2-Furoyl-pAph-Chg-PalMe(3)-NH₂;5-Me-thienyl-CO-pAph-Chg-PalMe(3)-NH₂, Ac-pAph-Chg-PalMe(3)-ol andTos-F(pNH₂)-Chg-R-L-P-NH₂.

EXAMPLE XXXIX Additional Factor Xa Inhibitors

The following compounds in Table 6 are Factor Xa Inhibitors with Ki<100μM. The compounds listed in Table 6 were confirmed by MS and AA.

TABLE 6 1. CF₃C(O)-(iBu)Phe(NH₂)-Chg-Arg-Leu-Pro-NH₂ 2.Ac-pAph-Chg-Arg-Pen(CH₂COOH)-Pro-NH₂ 3. Ac-pAph-Ile-Arg-Leu-Pro-NH₂ 4.Ac-pAph-Chg-Dab(CH ═N(CH₃)₂)-Leu-Pro-NH₂ 5.CF₃C(O)-(iBu)Nal(2)-Chg-Arg-Leu-Pro-NH₂ 6.Ac-Phe(3I,4NH₂)-Chg-Arg-Leu-Pro-NH₂ 7. CF₃C(O)-Tyr-Chg-Arg-Leu-Pro-NH₂(SEQ ID NO: 225) 8. (5-benzimidazoyl)-Phe(NH₂)-Chg-Arg-Leu-Pro-NH₂ 9.CF₃C(O)-(iBu)Tyr-Ile-Arg-Leu-Pro-NH₂ (SEQ ID NO: 226) 10.Ac-(Chx-CH₂)Tyr-Ile-Arg-Leu-Pro-NH₂ 11. D-Tyr-Chg-Arg-Leu-Pro-NH₂ 12.Ac-Trp-Chg-Arg-Leu-Pro-NH₂ 13. (2-benzofuroyl)-Tyr-Chg-Arg-Pen-Pro-NH₂14. (2-benzofuroyl)-pAph-Chg-PalMe(3)-Pen(CH₂COOH)-Pro-NH₂ 15.Ac-pAph-Chg-Arg-Cys(CH₂COOH)-Pro-NH₂ 16.(Alloc)-pAph-Chg-Arg-Leu-Pro-NH₂ 17.(2-benzofuroyl)-pAph-Chg-Arg-Pen(CH₂COOH)-Pro-NH₂ 18.Ac-pAph-Chg-PalMe(3)-Pen(CH₂COOH)-Pro-NH₂ 19.Ac-pAph-Chg-Arg-Leu-Pro-NH₂ 20. pAph-Chg-Arg-Leu-Pro-NH₂ 21.Ac-pAph-Chg-Arg-(HOOC—CH₂)Gly-Pro-NH₂ 22.Ac-pAph-Chg-Arg(HOOC—CH₂—CH₂)Gly-Pro-NH₂ 23. Ac-pAph-Chg-Arg-Gla-Pro-NH₂24. Ac-pAph-Chg-Arg-Cys(CH₂—COOH)-Pro-NH₂ 25.Ac-Pal(4)Me-Chg-Arg-Leu-Pro-NH₂ 26. Ac-(iBu)Nal(2)-Chg-Arg-Leu-Pro-NH₂27. Ac-Phe(p-CONH₂)-Chg-Arg-Leu-Pro-NH₂ 28.Ac-pAph-Chg-Arg-N[1(1,3-dicarboxy)propyl)]Gly-Pro-NH₂ 29.Ac-pAph-Chg-Dap(CH═N(CH₃)₂)-Leu-Pro-NH₂ 30.(2-quinolinoyl)-Phe(NH₂)-Chg-Arg-Leu-Pro-NH₂ 31.Ac-pAph-Chg-Arg-N(carboxymethyl)Gly-Pro-NH₂ 32.Ac-pAph-Chg-Arg-(carboxyethyl)Gly-Pro-NH₂ 33.Ac-mAph-Chg-Arg-Leu-Pro-NH₂ 34.Alloc-pAph-Chg-PalMe(3)-Pen(CH₂COOH)-Pro-NH₂ 35.Ac-pAph-Chg-Arg-N[1(1,3-dicarboxy)propyl)]Gly-Pro-NH₂ 36.Ac-pAph-Ile-Arg-Leu-Pro-NH₂ 37. Ac-Phe(pNH₂)-Chg-Arg-(Me)Leu-Pro-NH₂ 38.Ac-(Chx-CH₂)Tyr-Chg-Arg-Leu-Pro-NH₂ 39.(3-pyridoyl)-Phe(pNH₂)-Chg-Arg-Leu-Pro-NH₂ 40.(3-pyridoyl)-Nal(2)-Chg-Arg-Leu-Pro-NH₂ 41.Ac-Pal(4)Me-Chg-Pal(4)Me-Leu-Pro-NH₂ 42. Alloc-pAph-Chg-Arg-Leu-Pro-NH₂43. (4-isoquinolinoyl)-Phe(pNH₂)-Chg-Arg-Leu-Pro-NH₂ 44.Ac-pAph-Cha-PalMe(3)-(Me)Leu-Pro-NH₂ 45.Ac-pAph-Chg-PalMe(3)-Leu-Pro-NH₂ 46.(2-naphythl-CH₂)Phe(pNH₂)-Chg-Arg-Leu-Pro-NH₂ 47.(5-pyrazinoyl)Nal(2)-Chg-Arg-Leu-Pro-NH₂ 48.(Benzoyl)-Phe(pNH₂)-Chg-Arg-Leu-Pro-NH₂ 49.Ac-(2-methylpentanyl)-Tyr-Ile-Arg-Leu-Pro-NH₂ (SEQ ID NO: 227) 50.(2-pyridonyl)Phe(pNH₂)Chg-Arg-Leu-Pro-NH₂ 51.(Benzoyl)-Phe(pNH₂)-Chg-Arg-Leu-Pro-NH₂ 52.Ac-pAph-Chg-PalMe(3)-Leu-Pro-NH₂ 53.Ac-(2-methypentyl)Tyr-Ile-Arg-Leu-Pro-NH₂ (SEQ ID NO: 228) 54.Ac-(iBu)Phe(pCN)-Chg-Arg-Leu-Pro-NH₂ 55.Ac-(2-methybutyl)Tyr-Ile-Arg-Leu-Pro-NH₂ (SEQ ID NO: 229) 56.Ac-Phe(pNH₂)-Chg-Arg-Leu-Pro-NH₂ 57. Ac-Phe(pNH₂)-Chg-Arg-Leu-Hyp-NH₂58. Ac-Tyr-Chg-Arg-Leu-Pro-NH₂ 59.(2-naphthylsulfonyl)-Phe(pNH₂)-Chg-Arg-Leu-Pro-NH₂ 60.(2-methylbenzyl)-Phe(pNH₂)-Chg-Arg-Leu-Pro-NH₂ 61.(2-benzofuroyl)-Phe(pNH₂)-Chg-Dab(CH═N(CH₃)₂)- Leu-Pro-NH₂ 62.Ac-(cyclopentenyl-CH₂)Tyr-Ile-Arg-Leu-Pro-NH₂ (SEQ ID NO: 230) 63.Ac-Pal(4)Me-Chg-PalMe(3)-Leu-Pro-NH₂ 64.Ac-(iBu)-Phe(pNH₂)-Chg-Arg-Leu-Pro-NH₂ 65.Ac-(Chx-CH₂)-Tyr-Ile-Arg-Leu-Pro-NH₂ 66. Ac-pAph-Chg-Arg-Leu-NH₂ 67.Ac-pAph-Chg-Arg-Leu-OH 68. (2-benzofuroyl)-pAph-Chg-PalMe(3)-NH₂ 69.Ac-(iBu)Phe(pNH₂)-Chg-Arg-NH₂ 70. Alloc-pAph-Chg-PalMe(3)-NH₂ 71.(2-quinolinoyl)-pAph-Chg-PalMe(3)-NH₂ 72.Ac-pAph-Chg-PalMe(3)-NH(1-methoxycarbonyl)-1-cyclohexyl 73.Ac-pAph-Chg-Arg 74. (2-pyridoyl)-pAph-Chg-PalMe(3)-NH₂ 75.CF₃C(O)-(iBu)Phe(pNH₂)-Chg-Arg-NH₂ 76.Ac-pAph-Chg-PalMe(3)-NH-(1-methoxycarbonyl)-1-cyclopentyl 77.Ac-pAph-Chg-PalMe(3)-NH-(4-methoxycarbonyl- cyclohexyl)methyl 78.Ac-pAph-Chg-PalMe(3)-NH-(3-thienyl-2-carboxylic acid methyl ester) 79.Ac-pAph-Chg-Arg-NH₂ 80. CF₃C(O)-(iBu)Tyr-Chg-Arg-OH 81.Ac-pAph-Chg-PalMe(3)-NH-(4-methoxycarbonyl- cyclohexyl)methyl 82.Ac-pAph-Chg-PalMe(3)-NH₂ 83. Ac-pAph-Pgl-PalMe(3)-NH₂ 84.Ac-pAph-Chg-Pal(3)(CH₂COOH)-NH₂ 85. (2-quin)-pAph-Chg-PalMe(3)-NH₂ 86.Ac-pAph-Chg-PalMe(3)-NH-(4-carboxycyclohexyl) methyl 87.Ac-pAph-Chg-NH[4-(1-methyl-pyridinium)methyl] 88.(2-furoyl)-pAph-Chg-NH-(4-trimethyl-ammonium benzyl) 89.(3,4-dichlorobenzoyl)-pAph-Chg-NH-(4-trimethyl-ammonium benzyl) 90.(2-thienylacetyl)-pAph-Chg-NH-(4-trimethyl-ammonium benzyl) 91.(N-(5-methyl-2-thienoyl)-pAph-Chg-NH-(4-trimethyl-ammonium benzyl) 92.Ac-pAph-Chg-NH-(4-trimethyl-ammonium benzyl) 93.(Ethoxycarbonyl)-pAph-Chg-NH-(4-trimethyl-ammonium benzyl) 94.(2-fluorobenzoyl)-pAph-Chg-NH-(4-trimethyl-ammonium benzyl) 95.Ac-pAph-Chg-NH-(4-amidinobenzyl) 96.Alloc-pAph-Chg-NH-[4-(-methylpyridinium)-methyl] 97.(t-Butoxycarbonyl)-pAph-Chg-NH-(4-trimethyl-ammonium benzyl) 98.(2-furoyl)-pAph-Chg-NH-1-[3(N-methylpyridyl)]-1- (methylacetate)ethyl99. Ac-pAph-Chg-NH-1-[3(N-methylpyridyl)]-1-(methylacetate)ethyl 100.Ac-pAph-Chg-NH-[1-(1-methyl-4-pyridinium)ethyl 101.Ac-pAph-Chg-NH-[1-(1-methyl-4-pyridinium)methyl 102.Ac-pAph-Chg-NH-[1-(1-methyl-4-pyridinium)-2-hydroxy]ethyl 103.CF₃C(O)-(iBu)-Tyr-Ile-Arg-NH₂ 104. Ac-D-pAph-Chg-Arg-Leu-Pro-NH₂ 105.Ac-D-pAph-Chg-Arg-Gla-Pro-NH₂ 106.Ac-D-pAph-Chg-Arg-Cys-(CH₂—COOH)-Pro-NH₂ 107.Ac-D-pAph-Chg-Arg-N(carboxymethyl)Gly-Pro-NH₂ 108.Ac-D-pAph-Chg-Arg-(carboxyethyl)Gly-Pro-NH₂ 109.Ac-D-pAph-Chg-Arg-N[1(1,3-dicarboxy)propyl)]Gly-Pro-NH₂ 110.Ac-D-pAph-Ile-Arg-Leu-Pro-NH₂ 111. Alloc-D-pAph-Chg-Arg-Leu-Pro-NH₂ 112.Ac-D-pAph-Chg-PalMe(3)-Leu-Pro-NH₂ 113. Ac-D-pAph-Chg-Arg-NH₂.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

We claim:
 1. A compound selected from the group consisting ofCF₃C(O)-(iBu)Phe(NH₂)-Chg-Arg-Leu-Pro-NH₂; Ac-pAph-Ile-Arg-Leu-Pro-NH₂;CF₃C(O)-(iBu)Nal(2)-Chg-Arg-Leu-Pro-NH₂;Ac-Phe(3I,4NH₂)-Chg-Arg-Leu-Pro-NH₂; CF₃C(O)-Tyr-Chg-Arg-Leu-Pro-NH₂;(5-benzimidazoyl)-Phe(NH₂)-Chg-Arg-Leu-Pro-NH₂;CF₃C(O)-(iBu)Tyr-Ile-Arg-Leu-Pro-NH₂;Ac-(Chx-CH₂)Tyr-Ile-Arg-Leu-Pro-NH₂; D-Tyr-Chg-Arg-Leu-Pro-NH₂; andAc-Trp-Chg-Arg-Leu-Pro-NH₂.
 2. The compound selected from the groupconsisting of (2-benzofuroyl)-Tyr-Chg-Arg-Pen-Pro-NH₂;(2-benzofuroyl)-pAph-Chg-PalMe(3)-Pen(CH₂COOH)-Pro-NH₂;Ac-pAph-Chg-Arg-Cys(CH₂COOH)-Pro-NH₂; (Alloc)-pAph-Chg-Arg-Leu-Pro-NH₂;(2-benzofuroyl)-pAph-Chg-Arg-Pen(CH₂COOH)-Pro-NH₂;Ac-pAph-Chg-PalMe(3)-Pen(CH₂COOH)-Pro-NH₂; Ac-pAph-Chg-Arg-Leu-Pro-NH₂;Ac-pAph-Chg-Arg-(HOOC—CH₂)Gly-Pro-NH₂;Ac-pAph-Chg-Arg(HOOC—CH₂—CH₂)Gly-Pro-NH₂; Ac-pAph-Chg-Arg-Gla-Pro-NH₂;Ac-Pal(4)Me-Chg-Arg-Leu-Pro-NH₂; Ac-(iBu)Nal(2)-Chg-Arg-Leu-Pro-NH₂;Ac-Phe(pCONH₂)-Chg-Arg-Leu-Pro-NH₂;Ac-pAph-Chg-Arg-N[1(1,3-dicarboxy)propyl)]Gly-Pro-NH₂;Ac-pAph-Chg-Dap(CH═N(CH₃)₂)-Leu-Pro-NH₂;(2-quinolinoyl)-Phe(NH₂)-Chg-Arg-Leu-Pro-NH₂;Ac-pAph-Chg-Arg-N(carboxymethyl)Gly-Pro-NH₂;Ac-pAph-Chg-Arg-(carboxyethyl)Gly-Pro-NH₂; Ac-mAph-Chg-Arg-Leu-Pro-NH₂;Alloc-pAph-Chg-PalMe(3)-Pen(CH₂COOH)-Pro-NH₂;Ac-pAph-Ile-Arg-Leu-Pro-NH₂; Ac-Phe(pNH₂)-Chg-Arg-(Me)Leu-Pro-NH₂;Ac-(Chx-CH₂)Tyr-Chg-Arg-Leu-Pro-NH₂;(3-pyridoyl)-Phe(pNH₂)-Chg-Arg-Leu-Pro-NH₂;(3-pyridoyl)-Nal(2)-Chg-Arg-Leu-Pro-NH₂;Ac-Pal(4)Me-Chg-Pal(4)Me-Leu-Pro-NH₂;(4-isoquinolinoyl)-Phe(pNH₂)-Chg-Arg-Leu-Pro-NH₂;Ac-pAph-Chg-PalMe(3)-(Me)Leu-Pro-NH₂; Ac-pAph-Chg-PalMe(3)-Leu-Pro-NH₂;(2-naphythl-CH₂)Phe(pNH₂)-Chg-Arg-Leu-Pro-NH₂;(5-pyrazinoyl)Nal(2)-Chg-Arg-Leu-Pro-NH₂;(Benzoyl)-Phe(pNH₂)-Chg-Arg-Leu-Pro-NH₂;Ac-(2-methylpentanyl)-Tyr-Ile-Arg-Leu-Pro-NH₂;(2-pyridonyl)Phe(pNH₂)Chg-Arg-Leu-Pro-NH₂;Ac-(2-methypentyl)Tyr-Ile-Arg-Leu-Pro-NH₂;Ac-(iBu)Phe(pCN)-Chg-Arg-Leu-Pro-NH₂;Ac-(2-methybutyl)Tyr-Ile-Arg-Leu-Pro-NH₂;Ac-Phe(pNH₂)-Chg-Arg-Leu-Pro-NH₂; Ac-Phe(pNH₂)-Chg-Arg-Leu-Hyp-NH₂;Ac-Tyr-Chg-Arg-Leu-Pro-NH₂;(2-naphthylsulfonyl)-Phe(pNH₂)-Chg-Arg-Leu-Pro-NH₂;(2-methylbenzyl)-Phe(pNH₂)-Chg-Arg-Leu-Pro-NH₂;(2-benzofuroyl)-Phe(pNH₂)-Chg-Dab(CH═N(CH₃)₂)-Leu-Pro-NH₂;Ac-(cyclopentenyl-CH₂)Tyr-Ile-Arg-Leu-Pro-NH₂;Ac-Pal(4)Me-Chg-PalMe(3)-Leu-Pro-NH₂;Ac-(iBu)-Phe(pNH₂)-Chg-Arg-Leu-Pro-NH₂; andAc-(Chx-CH₂)-Tyr-Ile-Arg-Leu-Pro-NH₂.
 3. A compound selected from thegroup consisting of (2-benzofuroyl)-pAph-Chg-PalMe(3)-NH₂ andAc-(iBu)Phe(pNH₂)-Chg-Arg-NH₂.
 4. A compound selected from the groupconsisting of Alloc-pAph-Chg-PalMe(3)-NH₂;(2-quinolinoyl)-pAph-Chg-PalMe(3)-NH₂;Ac-pAph-Chg-PalMe(3)-NH(1-methoxycarbonyl)-1-cyclohexyl;Ac-pAph-Chg-Arg-NH₂; (2-pyridoyl)-pAph-Chg-PalMe(3)-NH₂;CF₃C(O)-(iBu)Phe(pNH₂)-Chg-Arg-NH₂;Ac-pAph-Chg-PalMe(3)-NH-(1-methoxycarbonyl)-1-cyclopentyl;Ac-pAph-Chg-PalMe(3)-NH-(4-methoxycarbonyl-cyclohexyl)methyl;Ac-pAph-Chg-PalMe(3)-NH-(3-thienyl-2-carboxylic acid methyl ester);CF₃C(O)-(iBu)Tyr-Chg-Arg-COOH; Ac-pAph-Chg-PalMe(3)-NH₂;Ac-pAph-Chg-Pal(3)(CH₂COOH)—NH₂;(2-quinolinecarboxy)-pAph-Chg-PalMe(3)-NH₂;Ac-pAph-Chg-PalMe(3)-NH-(4-carboxycyclohexyl)methyl; andCF₃C(O)(iBu)-Tyr-Ile-Arg-NH₂.
 5. A compound selected from the groupconsisting of Ac-D-pAph-Chg-Arg-Leu-Pro-NH₂;Ac-D-pAph-Chg-Arg-Gla-Pro-NH₂; Ac-D-pAph-Chg-Arg-Cys(CH₂—COOH)-Pro-NH₂;Ac-D-pAph-Chg-Arg-N(carboxymethyl)Gly-Pro-NH₂;Ac-D-pAph-Chg-Arg-(carboxyethyl)Gly-Pro-NH₂;Ac-D-pAph-Chg-Arg-N[1(1,3-dicarboxy)propyl)]Gly-Pro-NH₂;Ac-D-pAph-Ile-Arg-Leu-Pro-NH₂; Alloc-D-pAph-Chg-Arg-Leu-Pro-NH₂;Ac-D-pAph-Chg-PalMe(3)-Leu-Pro-NH₂; and Ac-D-pAph-Chg-Arg-NH₂.
 6. Acompound selected from the group consisting of Ac-pAph-Chg-Arg-Leu-NH₂and Ac-pAph-Chg-Arg-Leu.
 7. A compoundAc-D-pAph-Chg-PalMe(3)-Leu-Pro-NH₂.
 8. A compoundAc-D-pAph-Chg-PalMe(3)-NH₂.
 9. A compoundAc-Phe(pNH₂)-Chg-Arg-Leu-Pro-NH₂.
 10. A method of specificallyinhibiting the activity of Factor Xa, comprising contacting the factorXa with the compound as in claims 1, 2, 6, 3, 4, 5, 7, 8, or 9.